CN104349793A

CN104349793A - Oligonucleotide chelate complex-polypeptide compositions and methods

Info

Publication number: CN104349793A
Application number: CN201380026009.3A
Authority: CN
Inventors: 米歇尔·巴齐内; 安德鲁·瓦利恩特
Original assignee: Replicor Inc
Current assignee: Replicor Inc
Priority date: 2012-05-18
Filing date: 2013-05-17
Publication date: 2015-02-11
Anticipated expiration: 2033-05-17
Also published as: TW201408308A; TWI635864B; WO2013170386A1; HK1204279A1; CL2014003134A1; ZA201408674B; LT2849798T; ECSP14027694A; CO7131387A2; EA035967B1; MX346239B; KR20150013309A; CA2873529A1; IL235548B; CR20140527A; MY168778A; PH12014502551B1; JP2015517504A; PL2849798T3; DK2849798T3

Abstract

It is disclosed a pharmaceutical composition containing an oligonuclelotide chelate complex and at least one polypeptide or pegylated polypeptide. The present disclosure also describes additional pharmaceutical compositions and methods for the treatment of diseases including viral infections.

Description

Oligonucleotide chelate-peptide composition and method

The cross reference of related application

This application claims the priority of U.S. Provisional Application that the U.S. Provisional Application submitted on August 30th, 2012 number on May 18th, 61/695,035 and 2012 submits to numbers 61/648,711, their full content is incorporated herein by reference.

Technical field

This description relates to the compositions comprising oligonucleotide (ON) chelate and one or more not homopolypeptide or PEGylated polypeptides, for the preparation of comprising ON chelate and one or more the not method of the compositions of homopolypeptide or PEGylated polypeptides and the method by described compositions treatment various disease.

Background technology

Oligonucleotide (ON) chelate is intermolecular two or more ON connected by bivalence or other multivalent metal cation.With the intrinsic Chelating Properties using related side effects that may cause these compounds of ON in ON chelate.Using ON chelate is new method experimenter being used to ON, wherein alleviates relevant to non-chelated ON to use related side effects.These side effect may comprise with intravenous infusion shaking, generate heat and shivering with cold or injection site with subcutaneous administration hardens, inflammation and pain.In addition, by ON is prepared into chelate complexes, its pharmaco-kinetic properties may be improved, better curative properties can be provided with similar dosage compared with non-chelated ON.The characteristic sum character of ON chelate previously had description in international application published WO 2012/021985 and U.S. Application Publication number 2012/0046348, and described announcement in full way of reference is incorporated to herein.

ON chelate provides a kind of method using ON of improvement, and this method makes side effect reduce and not affect the biochemical activity of ON when using as simple sodium salt.

Comprise and to be correlated with by sequence or the ON chelate of ON that mechanism that sequence has nothing to do works can have therapeutic effect in disease state, it may carry out with described ON chelate not providing best therapeutic outcome in the deceased subject treated.

Therefore, need the new compositions providing a kind of improvement in the art, it comprises ON chelate, as in combination preparation.

Comprise and to be correlated with by sequence or the antiviral ON chelate of antiviral ON that mechanism that sequence has nothing to do works can infect different virus and have antivirus action.These antivirus actions may not provide optimum therapeuticing effect existing in the experimenter of viral infection of carrying out treating with described ON chelate.The therapeutic effect of further improvement can realize with the immunization therapy based on polypeptide with known antiviral activity by using antiviral ON chelate simultaneously.

Therefore, there is a need in the art for the new compositions of the combination comprising antiviral ON chelate and antiviral polypeptide or PEGylated polypeptides.Ideally, described antiviral polypeptide also has activity to discussed viral infection, by the impact target/biochemical route identical from discussed antiviral ON chelate or by affecting the different target/biochemical route affected with described antiviral ON chelate.

General introduction

According to this description, present providing package is containing the pharmaceutical composition of ON chelate and one or more not homopolypeptides.

According to this description, present providing package contains the anti-viral pharmaceutical compositions of antiviral ON chelate and one or more antiviral polypeptides or PEGylated polypeptides.

Disclose a kind of method of the disease therapy patient's condition, described method comprises the step experimenter of needs treatment being used to the pharmaceutical composition comprising ON chelate and polypeptide.

Disclose a kind of method of the disease therapy patient's condition, described method comprises the step by identical or different route of administration, the experimenter that needs are treated being used to independent pharmaceutical composition, and wherein the first compositions comprises ON chelate and the second compositions comprises polypeptide.

Disclose a kind of method for the treatment of viral infection, described method comprises the step experimenter of needs treatment being used to the pharmaceutical composition comprising antiviral ON chelate and antiviral polypeptide.

Disclose a kind of method for the treatment of viral infection, described method comprises the step by identical or different approach, the experimenter that needs are treated being used to independent pharmaceutical composition, and wherein one comprises antiviral ON chelate and another kind comprises antiviral polypeptide.

Disclose a kind of method for the treatment of viral infection, described method comprises the identical or different route of administration of use in same medicine compositions or the step using antiviral ON chelate and antiviral polypeptide in different pharmaceutical compositions.

The pharmaceutical composition openly comprising ON chelate and polypeptide is used for the treatment of the purposes of disease.

The pharmaceutical composition openly comprising ON chelate and polypeptide is manufacturing the purposes be used for the treatment of in the medicament of disease.

The first pharmaceutical composition openly comprising ON chelate and the second pharmaceutical composition comprising polypeptide are used for the treatment of the purposes of disease state, and described compositions is formulated for identical or different route of administration.

The pharmaceutical composition openly comprising antiviral ON chelate and antiviral polypeptide is used for the treatment of the purposes of viral infection.

Openly comprise antiviral ON chelate and antiviral polypeptide and manufacture the purposes be used for the treatment of in the medicament of viral infection.

Open pharmaceutical composition is used for the treatment of the purposes of viral infection, and one comprises antiviral ON chelate and another kind comprises antiviral polypeptide, and described pharmaceutical composition is formulated for be used respectively by identical or different approach.

Open antiviral ON chelate and antiviral polypeptide are used for the treatment of the purposes of viral infection, and described antiviral ON chelate and antiviral polypeptide are formulated in same medicine compositions or different pharmaceutical compositions for identical or different route of administration.

Providing package contains the pharmaceutical composition of ON chelate and at least one polypeptide be made up of two or more intermolecular oligonucleotide (ON) connected by bivalent cation.

A kind of pharmaceutical composition comprising antiviral ON chelate and at least one antiviral polypeptide be made up of two or more intermolecular antiviral ON connected by bivalent cation is provided.

In one embodiment, described antiviral polypeptide is by further Pegylation.

In another embodiment, described bivalent cation is the alkaline-earth metal with 2+ state of charge.

In another embodiment, described divalent metal is the transition metal with 2+ state of charge.

In another embodiment, described divalent metal is the lanthanide series metal with 2+ state of charge.

In another embodiment, described divalent metal is the late transition metal with 2+ state of charge.

In another embodiment, described divalent metal is calcium.

In another embodiment, described divalent metal is magnesium.

In another embodiment, described divalent metal is ferrum (2+), manganese, copper or zinc.

In another embodiment, described bivalent cation is made up of two or more different divalent metal.

In another embodiment, described bivalent cation is made up of calcium and magnesium.

In another embodiment, described ON chelate comprises at least one double-strand ON.

In another embodiment, described ON chelate comprises the ON that at least one has at least one phosphorothioate bond.

In another embodiment, described ON chelate comprises the ON of at least one complete phosphorothioate.

In another embodiment, described ON chelate comprises the ON that at least one has the ribose that one 2 ' is modified.

In another embodiment, described ON chelate comprises at least one each ribose by the methylated ON of 2 ' O-.

In another embodiment, described ON chelate comprises at least one ON containing at least one 5 ' methylcystein.

In another embodiment, described ON chelate comprise at least one wherein each cytosine be the ON of 5 ' methylcystein further.

In another embodiment, described ON chelate comprises the oligonucleotide being selected from SEQ ID NO:1-6 and 10-18.

In another embodiment, described ON chelate comprises the oligonucleotide being selected from SEQ ID NO:7-9.

In another embodiment, described polypeptide is following at least one:

Thymosin α1;

Any alpha-interferon or its polyethylene glycol derivative;

Any beta-interferon or its polyethylene glycol derivative;

Any gamma interferon or its polyethylene glycol derivative;

Any λ-interferon or its polyethylene glycol derivative;

Intederon Alpha-2a or α-2b or α-N3;

Interferon beta-1a or β-1b;

Gamma interferon 1-b;

Interferon lambda 1 or λ 2 or λ 3;

PEG ylated compound or α-2b or λ 1 or λ 2 or λ 3;

Myrcludex B；

Any antiviral cell factor or its polyethylene glycol derivative;

Thymus protein A; With

Any display has the polypeptide of antiviral activity or immunostimulatory activity.

In another embodiment, described pharmaceutical composition preparation is used for subcutaneous administration.

In another embodiment, described pharmaceutical composition preparation is used for intravenous infusion.

In another embodiment, the preparation of described pharmaceutical composition is used for the following route of administration of at least one: infusion in aerosol suctions, ophthalmic, oral, intestinal, intramuscular injection, peritoneal injection, intrathecal injection, sheath, tracheal strips, intravenous injection and locally.

In another embodiment, described ON chelate comprises the ON that at least one is made up of SEQ ID NO:2.

In another embodiment, described ON chelate comprises the ON that at least one is made up of SEQ ID NO:11.

In another embodiment, described ON chelate comprises the ON that at least one is made up of SEQ ID NO:18.

In another embodiment, described pharmaceutical composition comprises one or more following medicines further: Entecavir (entecavir), tenofovir disoproxil fumarate (tenofovir disoproxil fumarate), telbuvidine, adefovir ester (adefovir dipivoxil), lamivudine (lamivudine), ribavirin (ribavirin), VX-960 (telaprevir), Bo Saipuwei (boceprevir), GS-7977, tegobuvir, zanamivir (zanamivir), Oseltamivir (oseltamivir), ganciclovir (ganciclovir), FOSCARNET (foscarnet), acyclovir (acyclovir), azidothymidine AZT (zidovudine), Abacavir (abacavir), Lopinavir (lopinavir), ritonavir (ritonavir) or efavirenz (efavirenz).

In another embodiment, described pharmaceutical composition comprises pharmaceutically acceptable supporting agent further.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and PEG ylated compound that contain the oligonucleotide be made up of SEQ ID NO:3.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and PEG ylated compound that contain the oligonucleotide be made up of SEQ ID NO:18.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and PEG ylated compound that contain the oligonucleotide be made up of SEQ ID NO:11.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:3.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:18.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:11.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and Interferon Alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:3.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and Interferon Alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:18.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and Interferon Alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:11.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and Pegylation thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:3.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and Pegylation thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:18.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and Pegylation thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:11.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and glycol interferon alpha-2b. that contain the oligonucleotide be made up of SEQ ID NO:3

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and glycol interferon alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:18.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and glycol interferon alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:11.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and interferon lambda 1 that contain the oligonucleotide be made up of SEQ ID NO:3.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and interferon lambda 1 that contain the oligonucleotide be made up of SEQ ID NO:18.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and interferon lambda 1 that contain the oligonucleotide be made up of SEQ ID NO:11.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and glycol interferon λ 1 that contain the oligonucleotide be made up of SEQ ID NO:3.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and glycol interferon λ 1 that contain the oligonucleotide be made up of SEQ ID NO:18.

There is provided a kind of pharmaceutical composition, it comprises the ON chelate and glycol interferon λ 1 that contain the oligonucleotide be made up of SEQ ID NO:11.

There is provided a kind of method for the preparation of pharmaceutical composition described herein, described method comprises:

A. pharmaceutically at least one oligonucleotide (ON) sodium salt is dissolved in acceptable aqueous excipient;

B. in the ON of described dissolving, add pharmaceutically acceptable divalent metal saline solution gradually keep solvable to make described ON chelate;

C. pharmaceutically one or more antiviral polypeptides are dissolved in acceptable compatible aqueous excipient; And

D. described antiviral polypeptide solution and described ON chelate solution is made to mix gradually to make described ON chelate and antiviral polypeptide keep dissolving.

B. in the ON of described dissolving, add the pharmaceutically acceptable calcium of at least one and/or magnesium salt solution gradually keep solvable to make described ON chelate;

In one embodiment, described ON chelate solution and described polypeptide solution are facing the forward slip value using described pharmaceutical composition.

In another embodiment, the ratio of the divalent metal salt added in the ON of described dissolving is every 100mg oligonucleotide 0.1-50mg.

In another embodiment, final ON concentration is 0.1-200mg/ml.

In another embodiment, described divalent metal salt is at least one in chloride salt, gluconate, citrate, lactate, malate, aspartate, fumarate, Ascorbate, benzoate, erythorbate, propionate, sulfate or bicarbonate.

In another embodiment, described divalent metal saline solution comprises at least one in calcium, magnesium, ferrum (2+), manganese, copper or zinc.

A kind of test kit comprising pharmaceutical composition described herein is provided.

In one embodiment, described ON chelate and described at least one polypeptide are prepared respectively.

In another embodiment, described ON chelate and the preparation of described at least one polypeptide are for jointly using by identical or different route of administration.

Accompanying drawing is sketched

With reference now to accompanying drawing:

Fig. 1 illustrates the common physicochemical characteristics of ON.A) be jointly separated REP 2006 by high performance liquid chromatography and there are 21 mer phosphorothioate esterification ON of defined nucleotide sequence.B) material in described 21 aggressiveness ON is differentiated by mass spectrography.C) material in described REP2006ON is differentiated by mass spectrography.

Fig. 2 A illustrates the general chemical feature not relying on ON sequence of ON.Have nothing to do with sequence, any ON hydrophobicly to exist with the polymer of hydrophilic active as having simultaneously.The hydrophobicity that phosphorothioate (describing in chemical constitution in this drawing) is used for improving ON polymer does not still affect hydrophilic.Fig. 2 B schematically shows the ON Chelating Properties of bivalence and trivalent metal cation.Metal cation (being represented by the solid figure of Lycoperdon polymorphum Vitt) connects the hydrophilic surface of ON polymer by the metal ion bridge (being represented by ellipsis) between the non-bridge joint oxygen of two or three in phosphodiester bond or sulphur atom.

Fig. 3 illustrates the solution properties model of ON when there is bivalence or multivalent metal cation under different ON and divalent metal concentration.A) low divalent/trivalent metal cation, low ON concentration produces dimer or low order ON chelate.B) improve divalent/trivalent metal cation concentration and form ON chelate more completely in the solution.C) under bivalence or trivalent metal exist, improve ON concentration further can produce higher-order ON chelate along with metal concentration improves.Maintain dissolubility because hydrophilic surface is still exposed to aqueous environment, therefore (A) is to the equal water soluble solution of all chelates in (C).D) under enough ON and metal concentration, all hydrophilic surfaces are now limited in ON chelate, only make hydrophobic surface be exposed to aqueous environment.This causes the precipitation of ON chelate.

Fig. 4 illustrates that the solution properties of fluorescence-ON chelate is on the impact of fluorescence polarization.Along with metal concentration improves, the size (and quality) that ON chelate is formed also increases (see Fig. 3) and therefore rolls slower in the solution.Complex this slower rolling in the solution causes fluorescence polarization to raise and mP value raises.

Fig. 5 illustrates and uses the oligomer method (oligo method) described in EXAMPLE IV to be separated the HPLC that REP 2055 calcium chelates/Interferon Alpha-2b compositions is carried out.A small amount of not exclusively synthetic product (small arrow) normally has an ON of the sequence lacking one or more nucleotide compared with total length ON sequence prior to total length ON peak (large arrow) in chromatogram.This HPLC curve is typical for the whole non-purification ON (REP 2055, REP 2057 and REP2148) used.

Fig. 6 illustrates that the ESI-MS from the oligonucleotide content in the REP 2055 calcium chelates/Interferon Alpha-2b compositions at the HPLC peak of 16.70min in Fig. 5 analyzes.The quality of the main matter observed is 12612.2Da, determines that it is REP 2055 (expection m.wt.=12612.5).

Fig. 7 illustrates that the HPLC using method of protein 1 pair of REP 2055 calcium chelate/Interferon Alpha-2b compositions to carry out is separated (see EXAMPLE IV).

Fig. 8 illustrates that the ESI-MS from the peptide content in the REP 2055 calcium chelates/Interferon Alpha-2b compositions at the HPLC peak of 11.03min in Fig. 7 analyzes.The mass peak observed at 19263Da corresponds to Interferon Alpha-2b (expection m.wt.=19271Da), and corresponds to human albumin at the peak of 66560Da.

Fig. 9 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2057 calcium chelates/Interferon Alpha-2b compositions is carried out.As shown in Figure 5, a small amount of product not exclusively synthesized is prior to the main peak of total length REP 2057 sequence.

Figure 10 illustrates that the ESI-MS from the oligonucleotide content in the REP 2057 calcium chelates/Interferon Alpha-2b compositions at the HPLC peak of 15.46min in Fig. 9 analyzes.The quality of the main matter observed is 13413.5Da, determines that it is REP 2057 (expection m.wt.=13413.3).

Figure 11 illustrates that the HPLC using method of protein described herein 1 pair of REP 2057 calcium chelate/Interferon Alpha-2b compositions to carry out is separated (see EXAMPLE IV).

Figure 12 illustrates that the ESI-MS from the peptide content in the REP 2057 calcium chelates/Interferon Alpha-2b compositions at the HPLC peak of 11.43min in Figure 11 analyzes.The mass peak observed at 19264Da corresponds to Interferon Alpha-2b (expection m.wt.=19271Da), and corresponds to human albumin at the peak of 66618Da.

Figure 13 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2139 calcium chelates/Interferon Alpha-2b compositions is carried out.Do not find incomplete synthetic product.

Figure 14 illustrate from Figure 13 19.11 REP 2139 calcium chelates/Interferon Alpha-2b compositions in oligonucleotide content ESI-MS analyze.The quality of the main matter observed is 14095.3Da, determines that it is REP 2139 (expection m.wt.=14094.6).

Figure 15 illustrates that the HPLC using method of protein described herein 1 pair of REP 2139 calcium chelate/Interferon Alpha-2b compositions to carry out is separated (see EXAMPLE IV).

Figure 16 illustrates that the ESI-MS from the peptide content in the REP 2139 calcium chelates/Interferon Alpha-2b compositions at the HPLC peak of 11.12min in Figure 15 analyzes.The mass peak observed at 19264Da corresponds to Interferon Alpha-2b (expection m.wt.=19271Da), and corresponds to human albumin at the peak of 66674Da.

Figure 17 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2148 calcium chelates/Interferon Alpha-2b compositions is carried out.A small amount of not exclusively synthetic product is prior to the main peak of total length REP 2148 sequence.

Figure 18 illustrates that the ESI-MS from the oligonucleotide content in the REP 2148 calcium chelates/Interferon Alpha-2b compositions at the HPLC peak of 15.34min in Figure 17 analyzes.The quality of the main matter observed is 12891.6Da, determines that it is REP 2148 (expection m.wt.=12893.6).

Figure 19 illustrates that the HPLC using method of protein described herein 1 pair of REP 2148 calcium chelate/Interferon Alpha-2b compositions to carry out is separated (see EXAMPLE IV).

Figure 20 illustrates that the ESI-MS from the peptide content in the REP 2148 calcium chelates/Interferon Alpha-2b compositions at the HPLC peak of 8.44min in Figure 19 analyzes.The mass peak observed at 19265Da corresponds to Interferon Alpha-2b.

Figure 21 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2006 calcium chelates/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white arrow) at the peak of 6.31min eluting, and correspond to total length REP 2006 (black arrow) at the peak of 15.15min eluting.

Figure 22 illustrates that the ESI-MS from the oligonucleotide content in the REP 2006 calcium chelates/thymosin α1 compositions at the HPLC peak of 15.15min in Figure 21 analyzes.The quality observed is about 12650-13150Da, determines that it is REP 2006 (expection m.wt.=12612-13092Da).

Figure 23 illustrates that the ESI-MS from the peptide content in the REP 2006 calcium chelates/thymosin α1 compositions at the HPLC peak of 6.31min in Figure 21 analyzes.The quality of the main matter observed is 3108Da, determines that it is thymosin α1 (expection m.wt.=3108Da).

Figure 24 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2055 calcium chelates/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white) at the peak of 6.12min eluting, and correspond to total length REP 2055 (black arrow) at the peak of 16.70min eluting.

Figure 25 illustrates that the ESI-MS from the oligonucleotide content in the REP 2055 calcium chelates/thymosin α1 compositions at 16.70min HPLC peak in Figure 24 analyzes.The quality of the main matter observed is 12612.1Da, determines that it is REP 2055 (expection m.wt.=12612.5).

Figure 26 illustrates that the ESI-MS from the peptide content in the REP 2055 calcium chelates/thymosin α1 compositions at the HPLC peak of 6.12min in Figure 24 analyzes.The quality observing main matter is 3107.6Da, determines that it is thymosin α1 (m.wt.=3108Da).

Figure 27 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2057 calcium chelates/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white arrow) at the peak of 6.18min eluting, and correspond to total length REP 2057 (black arrow) at the peak of 15.47min eluting.

Figure 28 illustrates that the ESI-MS from the oligonucleotide content in the REP 2057 calcium chelates/thymosin α1 compositions at the HPLC peak of 15.47min in Figure 27 analyzes.The quality of the main matter observed is 13413.5Da, determines that it is REP 2057 (expection m.wt.=13413.3Da).

Figure 29 illustrates that the ESI-MS from the peptide content in the REP 2057 calcium chelates/thymosin α1 compositions at the HPLC peak of 6.18min in Figure 27 analyzes.The quality of the main matter observed is 3107.6Da, determines that it is thymosin α1 (expection m.wt.=3108Da).

Figure 30 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2139 calcium chelates/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white arrow) at the peak of 6.13min eluting, and correspond to total length REP 2139 (black arrow) at the peak of 19.01min eluting.

Figure 31 illustrates that-the ESIMS from the oligonucleotide content in the REP 2139 calcium chelates/thymosin α1 compositions at the HPLC peak of 19.01min in Figure 30 analyzes.The quality of the main matter observed is 14094.5Da, determines that it is REP 2139 (expection m.wt.=14094.6).

Figure 32 illustrates that the ESI-MS from the peptide content in the REP 2139 calcium chelates/thymosin α1 compositions at the HPLC peak of 6.13min in Figure 30 analyzes.The quality of the main matter observed is 3107.6Da, determines that it is thymosin α1 (expection m.wt.=3108Da).

Figure 33 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2148 calcium chelates/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white arrow) at the peak of 6.28min eluting, correspond to total length REP 2148 (black arrow) at the peak of 15.19min eluting.

Figure 34 illustrates that the ESI-MS from the oligonucleotide content in the REP 2148 calcium chelates/thymosin α1 compositions at the HPLC peak of 15.19min in Figure 33 analyzes.The quality of the main matter observed is 12892Da, determines that it is REP 2148 (expection m.wt.=12893Da).

Figure 35 illustrates that the ESI-MS from the peptide content in the REP 2148 calcium chelates/thymosin α1 compositions at the HPLC peak of 6.28min in Figure 33 analyzes.The quality of the main matter observed is 3107.3Da, determines that it is thymosin α1 (expection m.wt.=3108Da).

Figure 36 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2006 calcium chelates/PEG ylated compound compositions is carried out.

Figure 37 illustrates that the ESI-MS from the oligonucleotide content in the REP 2006 calcium chelates/PEG ylated compound compositions at the HPLC peak of 14.59min in Figure 36 analyzes.The quality of the main matter observed is about 12600-13200Da, determines that it is REP 2006 (expection m.wt.=12612-13092Da).

Figure 38 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2006 calcium chelates/PEG ylated compound compositions to carry out is separated (see EXAMPLE IV).

Figure 39 illustrates that the ESI-MS from the peptide content in the REP 2006 calcium chelates/PEG ylated compound compositions at the HPLC peak of 13.21min in Figure 38 analyzes.

Figure 40 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2055 calcium chelates/PEG ylated compound compositions is carried out.

Figure 41 illustrates that the ESI-MS from the oligonucleotide content in the REP 2055 calcium chelates/PEG ylated compound compositions at the HPLC peak of 16.71min in Figure 40 analyzes.The quality of the main matter observed is 12612.4Da, determines that it is REP 2055 (expection m.wt.=12612.5Da).

Figure 42 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2055 calcium chelates/PEG ylated compound compositions to carry out is separated (see EXAMPLE IV).

Figure 43 illustrates that the ESI-MS from the peptide content in the REP 2055 calcium chelates/PEG ylated compound compositions at the HPLC peak of 8.03min in 42 figure analyzes.

Figure 44 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2057 calcium chelates/PEG ylated compound compositions is carried out.

Figure 45 illustrates that the ESI-MS from the oligonucleotide content in the REP 2057 calcium chelates/PEG ylated compound compositions at the HPLC peak of 15.45min in Figure 44 analyzes.The quality of the main matter observed is 13413.5Da, determines that it is REP 2057 (expection m.wt.=13413.3Da).

Figure 46 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2057 calcium chelates/PEG ylated compound compositions to carry out is separated (see EXAMPLE IV).

Figure 47 illustrates that the ESI-MS from the peptide content in the REP 2057 calcium chelates/PEG ylated compound compositions at the HPLC peak of 8.04min in Figure 46 analyzes.

Figure 48 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2139 calcium chelates/PEG ylated compound compositions is carried out.

Figure 49 illustrates that the ESI-MS from the oligonucleotide content in the REP 2139 calcium chelates/PEG ylated compound compositions at the HPLC peak of 19.08min in Figure 48 analyzes.The quality of the main matter observed is 14095.3Da, determines that it is REP 2139 (expection m.wt.=14094.6Da).

Figure 50 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2139 calcium chelates/PEG ylated compound compositions to carry out is separated (see EXAMPLE IV).

Figure 51 illustrates that the ESI-MS from the peptide content in the REP 2139 calcium chelates/PEG ylated compound compositions at the HPLC peak of 8.02min in Figure 50 analyzes.

Figure 52 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2148 calcium chelates/PEG ylated compound compositions is carried out.

Figure 53 illustrates that the ESI-MS from the oligonucleotide content in the REP 2148 calcium chelates/PEG ylated compound compositions at the HPLC peak of 15.42min in Figure 52 analyzes.The quality of the main matter observed is 12891.5Da, determines that it is REP 2148 (expection m.wt.=12893Da).

Figure 54 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2148 calcium chelates/PEG ylated compound compositions to carry out is separated (see EXAMPLE IV).

Figure 55 illustrates that the ESI-MS from the peptide content in the REP 2148 calcium chelates/PEG ylated compound compositions at the HPLC peak of 13.17min in Figure 54 analyzes.

Figure 56 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2055 calcium chelates/interferon lambda 1 compositions is carried out.

Figure 57 illustrates that the ESI-MS from the oligonucleotide content in REP 2055 calcium chelates/interferon lambda 1 compositions at the HPLC peak of 14.84min in Figure 56 analyzes.The quality of the main matter observed is 12610.6Da, determines that it is REP 2055 (expection m.wt.=12612.5Da).

Figure 58 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2055 calcium chelates/interferon lambda 1 compositionss to carry out is separated (see EXAMPLE IV).

Figure 59 illustrates that the ESI-MS from the peptide content in REP 2055 calcium chelates/interferon lambda 1 compositions at the HPLC peak of 10.68min in Figure 58 analyzes.The primary front observed is at 20139Da, consistent with the approximate molecular weight of interferon lambda 1.

Figure 60 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2139 calcium chelates/interferon lambda 1 compositions is carried out.

Figure 61 illustrates that the ESI-MS from the oligonucleotide content in REP 2139 calcium chelates/interferon lambda 1 compositions at the HPLC peak of 17.31min in Figure 60 analyzes.The quality of the main matter observed is 14095.3Da, determines that it is REP 2139 (expection m.wt.=14094.6Da).

Figure 62 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2139 calcium chelates/interferon lambda 1 compositionss to carry out is separated (see EXAMPLE IV).

Figure 63 illustrates that the ESI-MS from the protein content in REP 2139 calcium chelates/interferon lambda 1 compositions at the HPLC peak of 10.53min in Figure 62 analyzes.The primary front observed is at 20139Da, consistent with the approximate molecular weight of interferon lambda 1

Figure 64 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2148 calcium chelates/interferon lambda 1 compositions is carried out.

Figure 65 illustrates that the ESI-MS from the oligonucleotide content in REP 2148 calcium chelates/interferon lambda 1 compositions at the HPLC peak of 15.47min in Figure 64 analyzes.The quality of the main matter observed is 12891.8Da, determines that it is REP 2148 (expection m.wt.=12893Da).

Figure 66 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2148 calcium chelates/interferon lambda 1 compositionss to carry out is separated (see EXAMPLE IV).

Figure 67 illustrates that the ESI-MS from the protein content in REP 2148 calcium chelates/interferon lambda 1 compositions at the HPLC peak of 10.51min in Figure 66 analyzes.The primary front observed is at 20139Da, consistent with the approximate molecular weight of interferon lambda 1

Figure 68 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2055 magnesium chelate/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white arrow) at the peak of 5.86min eluting, and correspond to total length REP 2055 (black arrow) at the peak of 14.62min eluting.

Figure 69 illustrates that the ESI-MS from the oligonucleotide content in the REP 2055 magnesium chelate/thymosin α1 compositions at the HPLC peak of 14.62min in Figure 68 analyzes.The quality of the main matter observed is 12610.1Da, determines that it is REP 2055 (expection m.wt.=12612.5Da).

Figure 70 illustrates that the ESI-MS from the peptide content in the REP 2055 magnesium chelate/thymosin α1 compositions at the HPLC peak of 5.82min in Figure 68 analyzes.The quality of the main matter observed is 3107.1Da, determines that it is thymosin α1 (expection m.wt.=3108Da).

Figure 71 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2055 calcium mixtures-magnesium chelate/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white arrow) at the peak of 6.02min eluting, and correspond to total length REP 2055 (black arrow) at the peak of 14.85min eluting.

Figure 72 illustrates that the ESI-MS from the oligonucleotide content in the REP 2055 calcium mixtures-magnesium chelate/thymosin α1 compositions at the HPLC peak of 14.85min in Figure 71 analyzes.The quality of the main matter observed is 12609.7Da, determines that it is REP 2055 (expection m.wt.=12612.5Da).

Figure 73 illustrates that the ESI-MS from the peptide content in the REP 2055 calcium mixture magnesium chelate/thymosin α1 compositions at the HPLC peak of 6.02min in Figure 71 analyzes.The quality of the main matter observed is 3107.1Da, determines that it is thymosin α1 (expection m.wt.=3108Da).

Figure 74 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2055 magnesium chelate/PEG ylated compound compositions is carried out.

Figure 75 illustrates that the ESI-MS from the oligonucleotide content in the REP 2055 magnesium chelate/PEG ylated compound compositions at the HPLC peak of 14.83min in Figure 74 analyzes.The quality of the main matter observed is 12610.2Da, determines that it is REP 2055 (expection m.wt.=12612.5Da).

Figure 76 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2055 magnesium chelate/PEG ylated compound compositionss to carry out is separated (see EXAMPLE IV).

Figure 77 illustrates that the ESI-MS from the peptide content in the REP 2055 magnesium chelate/PEG ylated compound compositions at the HPLC peak of 13.44min in Figure 76 analyzes.

Figure 78 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2055 calcium mixtures-magnesium chelate/PEG ylated compound compositions is carried out.

Figure 79 illustrates that the ESI-MS from the oligonucleotide content in the REP 2055 calcium mixtures-magnesium chelate/PEG ylated compound compositions at the HPLC peak of 14.83min in Figure 78 analyzes.The quality of the main matter observed is 12609.8Da, determines that it is REP2055 (expection m.wt.=12612.5Da).

Figure 80 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2055 calcium mixtures-magnesium chelate/PEG ylated compound compositions to carry out is separated (see EXAMPLE IV).

Figure 81 illustrates that the ESI-MS from the peptide content in the REP 2055 calcium mixtures-magnesium chelate/PEG ylated compound compositions at the HPLC peak of 13.44min in Figure 80 analyzes.

Figure 82 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2139 magnesium chelate/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white arrow) at the peak of 5.84min eluting, and correspond to total length REP 2139 (black arrow) at the peak of 16.86min eluting.

Figure 83 illustrates that the ESI-MS from the oligonucleotide content in the REP 2139 magnesium chelate/thymosin α1 compositions at the HPLC peak of 16.86min in Figure 82 analyzes.The quality of the main matter observed is 14091.3Da, determines that it is REP 2139 (expection m.wt.=14094.6Da).

Figure 84 illustrates that the ESI-MS from the peptide content in the REP 2139 magnesium chelate/thymosin α1 compositions at the HPLC peak of 5.84min in Figure 82 analyzes.The quality of the main matter observed is 3107.1Da, determines that it is thymosin α1 (expection m.wt.=3108Da).

Figure 85 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2139 calcium mixtures-magnesium chelate/thymosin α1 compositions is carried out.Correspond to thymosin α1 (white arrow) at the peak of 5.84min eluting, and correspond to total length REP 2139 (black arrow) at the peak of 16.61min eluting.

Figure 86 illustrates that the ESI-MS from the oligonucleotide content in the REP 2139 calcium mixtures-magnesium chelate/thymosin α1 compositions at the HPLC peak of 16.61min in Figure 85 analyzes.The quality of the main matter observed is 14091.1Da, determines that it is REP 2139 (expection m.wt.=14094.6Da).

Figure 87 illustrates that the ESI-MS from the peptide content in the REP 2139 calcium mixture magnesium chelate/thymosin α1 compositions at the HPLC peak of 5.84min in Figure 85 analyzes.The quality of the main matter observed is 3107.1Da, determines that it is thymosin α1 (expection m.wt.=3108Da).

Figure 88 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2139 magnesium chelate/PEG ylated compound compositions is carried out.

Figure 89 illustrates that the ESI-MS from the oligonucleotide content in the REP 2139 magnesium chelate/PEG ylated compound compositions at the HPLC peak of 17.19min in Figure 88 analyzes.The quality of the main matter observed is 14091.5Da, determines that it is REP 2139 (expection m.wt.=14094.6Da).

Figure 90 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2139 magnesium chelate/PEG ylated compound compositionss to carry out is separated (see EXAMPLE IV).

Figure 91 illustrates that the ESI-MS from the peptide content in the REP 2139 magnesium chelate/PEG ylated compound compositions at the HPLC peak of 13.38min in Figure 90 analyzes.

Figure 92 illustrates and uses oligomer method described herein to be separated (see EXAMPLE IV) to the HPLC that REP 2139 calcium mixtures-magnesium chelate/PEG ylated compound compositions is carried out.

Figure 93 illustrates that the ESI-MS from the oligonucleotide content in the REP 2139 calcium mixtures-magnesium chelate/PEG ylated compound compositions at the HPLC peak of 16.95min in Figure 92 analyzes.The quality of the main matter observed is 14091.4Da, determines that it is REP2139 (expection m.wt.=14094.6Da).

Figure 94 illustrates that the HPLC using method of protein described herein 2 pairs of REP 2139 calcium mixtures-magnesium chelate/PEG ylated compound compositions to carry out is separated (see EXAMPLE IV).

Figure 95 illustrates that the ESI-MS from the peptide content in the REP 2139 calcium mixtures-magnesium chelate/PEG ylated compound compositions at the HPLC peak of 13.46min in Figure 94 analyzes.

Detailed Description Of The Invention

As described in international application published WO 2012/021995 and U.S. Application Publication number 2012/0046348 (as described in announcement content in full way of reference be incorporated to herein), ON containing any bivalence simple metal cation (such as but not limited to, Ca ²⁺, Mg ²⁺and Fe ²⁺) aqueous solution in do not exist in a salt form, but to exist with the complex form of the chelating of ON.These complex are made up of ON dimer or high-order molecular organization, and wherein ON is by the di-phosphate ester of bivalent metal ion bridge at them or (see Fig. 2 B) of phosphorothioate backbone moleculartie.Under specific ON and metal cation concentration, the complex of these chelatings is stable and solvable in aqueous, and any bivalent cation of effectively isolating in ON chelate and solution interact.This chelate is formed also may there is (describing in as Fig. 2 B) in the simple metal cation situation with 3+ electric charge or higher electric charge.Therefore ON plays multivalent metal cation chelating agen (chelater) and does not form salt with multivalent metal cation.

ON chelate can contain various multivalent metal cation, comprises calcium, magnesium, cobalt, ferrum, manganese, barium, nickel, copper, zinc, cadmium, hydrargyrum and lead.The chelating of further these multivalent metal cations of display causes being formed the ON chelate that consists of the ON that metal cation connects two or more and occurs when length is greater than the ON of 6 nucleotide and under the existence of ON with di-phosphate ester or phosphorothioate bond.ON can optional each key by phosphorothioate.Chelating is also to occur when ribose modifies (as 2 ' O methyl) or the ON containing the such as modified base such as 5 ' methylcystein or 4-paper substrate containing 2 '.These 2 ' are modified and to may reside on one or more or all ribose and the base of modifying may reside in one or more base or is prevalent in (namely all cytosine exist with the form of 5 ' methylcystein) in each base.In addition, ON chelate can comprise containing multiple modification as each key is modified and the adorned ON of each base by 2 ' by phosphorothioate, each ribose.Hereafter further definition and ON chelate form compatible ON and modify.In addition, the chelating of metal cation and the nucleotide sequence of existence have nothing to do, but depend on the common physicochemical characteristics of all ON (see Fig. 2 A).

Although the formation of ON chelate can realize with any divalent metal, but intention be used as medicine ON chelate should preferably only containing calcium and or magnesium, but also can contain the ferrum of trace, manganese, copper or zinc, and should not comprise cobalt, barium, nickel, cadmium, hydrargyrum, lead or any other unlisted divalent metals here.

Importantly, the formation of ON chelate does not occur in such as Na ⁺, K ⁺or NH ₄ ⁺deng therefore univalent cation can not occur on any univalent cation.Therefore, term " ON salt " be only limitted to more accurately ON and univalent cation or with the cationic salt or not forming chelate ON.

In ON known at least partially and blood, the instantaneous interaction of protein component may be mediated by ON and the interaction of the such as calbindin such as albumin and Ca-dependent coagulation cascade albumen.Therefore use ON (this significantly reduces or eliminates their tendencies interactional with calbindin) with the complex form of chelating will reduce the interaction in blood of these protein and make the side effect (as instantaneous anticoagulation) of using with ON less and the ON dose number arriving target organ (such as liver, lung or spleen) can also be improved compared with non-chelated ON.

Fluorescence polarization is the common methods for studying intermolecular interaction.In this technology, with fluorescent marker (such as FITC) labelling determinand (i.e. any ON).In the solution, determinand molecule is free to tumble in the solution due to Brownian movement, and when this makes the optical excitation determinand with appropriate wavelength, polarized fluorescence is launched very weak.Use the part of enough molecular weight (at least the same with determinand size), the interaction between determinand and part brings the substance to described complex rolls in the solution to suppress.Owing to inhibit this rolling in solution, therefore when exciting fluorescent emission by remarkable polarization.Therefore use this technology, can measure in the solution and interact and all physical restriction is not had to any one binding partners.Fluorescence polarization is reported as nondimensional mP, and it is directly proportional to the mark of the conjunction type determinand molecule in reaction.Such as, if the determinand molecule of very little ratio is combined by particular ligand, so fluorescence polarization will seldom and therefore mP value is also little.Another is extreme, if most of determinand molecule is combined the part of higher concentration (or with) by particular ligand, will have sizable fluorescence polarization and therefore mP value is also large.In this way, can by change under the existence of the fluorescent labeling determinand of fixed amount ligand concentration produce particular test-ligand interaction in conjunction with isopotential line.

Use herein different fluorescent labeling ON to study they multivalent metal cation exist under complex formed.Although being formed by fluorescence polarization monitoring complex needs these ON to be fluorescently labeled, this labelling is fixed on the 3 ' end of ON, not disturb nitrogenous base or the phosphodiester backbone of discussed ON.In addition, in order to get rid of any disturbance of the normal ON behavior in solution further, fluorescent marker is kept away from by 3 carbon joints of rigidity and ON.Therefore any ON complex formation using fluorescence polarization fluorescent labeling ON to observe herein is all the accurate performance of unmarked ON (no matter whether forming complex) solution behavior.

Standard in this area clearly teaches the way to needing the experimenter for the treatment of to use ON with ON sodium salt.This was demonstrated by the ON using numerous sodium-salt form in clinical trial, they comprise Fomivirisen (ISIS 2922), Mipomersen (ISiS 301012), Trecovirsen (GEM 91), Custirsen (OGX-011/ISIS 112989), Genasense (G3139), Aprinocarsen (ISIS 3531/LY 900003), PRO-51 (GSK 2402968) and ALN-RSV01 (Geary etc., 2002, Clin.Pharmacokinetics, 41:255-260; Yu etc., 2009, Clin.Pharmacokinetics, 48:39-50; Sereni etc., 1999, J.Clin.Pharmacol., 39:47-54; Chi etc., 2005, J.Nat.Canc.Inst., 97:1287-1296; Marshall etc., 2004, Ann.Oncol., 15:1274-1283; Grossman etc., 2004, Neuro-Oncol, 6:32-40; Goemans etc., 2011NEJM 364:1513-1522).Not announcing instruction at present uses the preparation of calcium, magnesium or other divalent metal for the data of the oligonucleotide of parenteral administration.

Can owing to their chelating effect with relevant many side effect of using of sodium salt ON.The anticoagulation of ON is weakened Ca-dependent coagulation cascade by serum calcium cause by ON chelating at least partly.This chelating of serum calcium and its potential serum low blood calcium that can cause are also consistent with being used the side effect that ON observes by IV, and these side effect comprise heating, shake, weak and arteriotony declines (the latter is with quick IV infusion or injection).The injection site reaction (sclerosis, inflammation, fragility and pain) observed with subcutaneous injection ON is because the calcium in injection site and other possible bivalence or polyvalent cation (as magnesium) are by ON locally chelating at least partly.Use with the complex form of chelating that ON is verified alleviates these side effect many.

In addition, because all ON chelate can be formed with divalent metal and any ON in the solution, so by the exemplary oligonucleotide sequence that uses degeneracy ON REP 2006 and modify with different ON [in the examples below (AG) ₂₀(AC) ₂₀] multiple polypeptides in solution and PEGylated polypeptides and any formation of ON chelate and the instruction of the compatibility will be proved amply to those skilled in the art.The stable formation of the compositions of the above ON material containing the chelate from different polypeptide or PEGylated polypeptides will prove that any ON chelate (function specific with it has nothing to do) person can form stabilizing solution with multiple polypeptides or PEGylated polypeptides, this will be desirable for the treatment of specific disease indication (such as glycol interferon is used for the treatment of hepatitis B or hepatitis C, or myrcludex B is used for the treatment of hepatitis B) in the art.

Term oligonucleotide (ON) refers to oligomer or the polymer of ribonucleic acid (RNA) and/or DNA (deoxyribonucleic acid) (DNA).This term comprises by the core base of modifying (comprising 5 ' methylcystein and 4 ' paper substrate), the ON that forms of (skeleton) binding and have the ON of part of functionally similar non-natural existence between sugar and covalency nucleoside.Due to desired characteristic, the stability under the existence of nuclease to nucleic acid target target affinity and increase of the immunoreactivity such as reduced, the cellular uptake of enhancing, enhancing, the ON of this kind of modification or replacement may be more preferred than native form.ON also can be double-strand.

ON in the disclosure can comprise various modification, and such as stabilisation is modified, and therefore at phosphodiester bond and/or can comprise at least one modification on sugar and/or in base.Such as, ON can include, but is not limited to one or more modification, or be fully modified so that containing having all keys of described modification or sugared or base, the key of modification can comprise phosphorothioate bond, phosphordithiic acid ester bond and/or methylphosphonic acid ester bond.Although the key modified is useful, ON can comprise phosphodiester bond.Other useful modification includes, but is not limited to the modification of the 2 ' position at sugar, comprise 2 '-O-alkyl modified, as 2 '-O-methyl modification, 2 ' O-methoxy ethyl (2'MOE), 2 '-amido modified, 2 '-halo modification, as 2 '-fluorine; Acyclonucleosides acid-like substance.Other 2 ' modifications are also known in the art and can use, as lock nucleic acid.Specifically, ON has the key of modification everywhere or each key is modified, such as, and phosphorothioate; There is 3 '-and/or 5 '-medicated cap; Comprise end 3 '-5 ' key; Described ON is or comprises the concatemer be made up of two or more ON connected by joint.Base modification can comprise 5 ' of cytosine base and to methylate (5 ' methylcystein or in the environment of nucleotide, 5 ' methylcytidine) and/or the 4 ' sulfuration (4 ' deracil or in the environment of nucleotide, 4 ' thio uridine) of uracil base.When synthesis condition is when chemically compatible, modifying (as 2 ' O-methylates) and modified base (as 5 ' methylcystein) as having with phosphorothioate bond, 2 ' ribose, the key of the compatible modification of different chemical can be merged.ON can be modified completely (such as further by all these different modifying, each key phosphorothioate, each ribose is modified by 2 ' O methyl and each cytosine base has 5 ' methyl modification (5 ' methylcystein) in addition).

In this manual, term " antiviral ON " refers to and directly or indirectly can suppress some aspects of virus replication by means of its specific biochemical activity (being no matter that sequence is correlated with or sequence has nothing to do) or directly or indirectly strengthen host by immunity or other machine-processed any ON removing the ability infected.

In the disclosure, term " ON chelate " refers to the complex of intermolecular two or more ON connected by multivalent metal cation in the solution.

In the disclosure, term " antiviral ON chelate " refers to the complex of intermolecular two or more antiviral ON connected by multivalent metal cation in the solution.Described antiviral ON chelate can be made up of the antiviral ON of single kind or two or more different types of antiviral ON, they may have identical or different mechanism of action (such as, two or more antisense ON, at least one antisense ON and at least one is fit, at least one antisense ON and at least one siRNA).

In the disclosure, term " antiviral polypeptide " refers to and directly or indirectly can suppress some aspects of virus replication by means of its specific biochemical activity (being no matter that sequence is correlated with or sequence has nothing to do) or directly or indirectly strengthen host by immunity or other machine-processed polypeptide removing the ability infected.Described polypeptide can be natural acquisition or restructuring.Described polypeptide can from a part natural exist polypeptide restructuring obtain.Described polypeptide can be Pegylation or non-Pegylation.

In the disclosure, term " degeneracy ON " means the strand ON in each position with swing (N), as NNNNNNNNNN.Each base synthesizes wobble base, exists using the sequence that the different random making this ON in fact have equal length and physicochemical property as a group generates.Such as, be the degeneracy ON of 40 bases for length, any particular sequence in group only represents 1/4 of gross score in theory ⁴⁰or 8.3X 10 ^-25.Assuming that 1 mole=6.022X10 ²³individual molecule, and in fact the quality of 1 mole of 40 aggressiveness ON is approximately 12-14kg (depending on sequence and the modification of existence), and in fact any ON with effective particular sequence occurs being no more than once in any preparation.Therefore any chelate formation observed in this preparation or biological activity must be the physicochemical properties of the non-sequence relevant (or haveing nothing to do with described sequence) due to ON, because can not expect that any specific ON (being unique in the described preparation) contribution sources with defined nucleotide sequence is in any activity of its specific nucleotide sequence.

As the Still another example of this concept, example I compares REP 2006 (having 40 aggressiveness ON of the sequence of the complete phosphorothioate of degeneracy) and the sign of 21 aggressiveness ON with defined nucleotide sequence (also completely by phosphorothioate) by high pressure lipuid chromatography (HPLC) and mass spectrography, and clearly display any ON with similar size and chemical modification (i.e. phosphorothioate) will have similar (if the not identical) physicochemical characteristics (see Figure 1A-C) of height that the nucleotide sequence that do not existed affects.

In this application, term " nucleic acid polymers " or NAP are intended to any strand ON being used for differentiating not comprise sequence specific sexual function.The biochemical activity of NAP do not rely on ON Toll-like receptor identification, derive from the fit interaction of the specific secondary/tri-grade ON structure of the particular order of the nucleotide of existence with the hybridization of target nucleic acid or needs.NAP can comprise base as above and or key and or sugar-modified.

Numerous mechanism that ON can be correlated with by sequence or sequence has nothing to do play their therapeutical effect.The mechanism that sequence is correlated with is that it active needs specific nucleotide sequence and those mechanism of reducing because of the one or more change in the nucleotide sequence that exists of wherein said activity.This specific sequence may contain the whole length of ON or only have its part (sequence motifs).The example of the ON that sequence is correlated with comprises:

1. antisense ON (strand or double-strand (such as, siRNA (siRNA) or children purpura nephritis (shRNA)) be with the specific part complementation of studied messenger RNA (mRNA) and when introducing in cell, reticent complex (RISC) degraded that they guide specific mRNA to be induced by RNA enzyme H or RNA.

2. steric block (Stearic blocking) ON is with the specific part complementation of mRNA but by the engineered single-stranded antisense ON becoming not activate RNA enzyme H.These ON and their target mRNA hybridize the double stranded section producing and the protein usually acting on described mRNA is provided to steric hindrance (stearic hindrance).These ON may be used for blocking the translation of specific mRNA or specific mRNA's transcribe rear montage and maturation for disturbing.These ON can be blocked RNA enzyme H by engineered one-tenth by complete 2 ' ribose modification (as 2 ' O methylates) and activate (because the activation of RNA enzyme H and the mechanism of action of these ON are not an entirety).

3. fit be take to carry out the interactional specific three-dimensional conformation of specific protein and not easily with host DNA or the interactional ON of RNA.Fitly can also comprise mirror image isomer, their use L-nucleotide substitution D-nucleotide to give height enzyme stability to described ON.

4. the specific 6 aggressiveness nucleic acid motifs (XXCGXX) of immunostimulating ON use stimulate the immunne response in mammal.Best motif changes with the change of species, but strictly depends on the particular sequence consistent with XXCGXX motif.

5. micro rna (miRNA) combines and blocks the natural function that there is micro rna molecule involved in the adjustment of various biochemical route.

The example of the ON that the sequence of Unique notification has nothing to do is phosphorothioate NAP, it by means of its physicochemical property as amphipathic polymer with the relevant way selection ground of size (length) and amphipathic proteinaceous matter structural interaction.

The current approved of some antiviral drugs based on polypeptide is used for the treatment of viral infection, and they comprise PEG ylated compound (being used for the treatment of hepatitis B (HBV) and hepatitis C (HCV)), Interferon Alpha-2b (being used for the treatment of HBV), thymosin α1 (being used for the treatment of HBV) and enfurtide (being used for the treatment of HIV-1).Other medicine based on polypeptide is also had to develop, comprise myristyl pre-s1HBV surface antigen protein fragment (the myrcludex B being used for the treatment of HBV, Petersen etc., 2008, Nature Biotech.26:335-341) and glycol interferon λ 1 (Muir etc., 2010Hepatology 52:822-832).In addition, interferon lambda 1, λ 2 and λ 3 are also known has antiviral activity (Friborg etc., 2013, Antimicrobial Agents and Chemotherapy 57:1312-1322).When Pegylation or non-glycol interferon and other known immunomodulator (such as thymosin α1), these polypeptide have the activity stimulating its corresponding biochemical route, and this stimulation causes the stimulation of the immunne response to experimenter's viral infection.Although these polypeptide can successfully be replied by immune stimulatory, the patient using these medicines based on polypeptide to only have fraction to suffer from HBV or HCV infection realizes its control completely infected.Myrcludex B enters hepatocyte for blocking HBV, but its treatment benefit needs to prove in people patient.

Except these polypeptide, also have the polypeptide with known antiviral activity of other kind, they can be used for when combining with ON chelate treating viral infection.These polypeptide comprise cytokine, such as, but not limited to TNF-α, IL-1 β, IL-2, IL-4, IL-6 and interferon gamma.

For the compatibility of Pegylation is carried out to therapeutic activity polypeptide method and Pegylation and Peptides activity in this area be know and comprise particular amino acid residue Polyethylene Glycol (PEG) chain being connected to discussed polypeptide.The major function of Pegylation is the cycle life of increase polypeptide and also makes its immunogenicity reduce.These features improve the toleration of the polypeptide discussed and the administration frequency reduced required for optimum therapeuticing effect.This area further known can realize PEG residue and polypeptide connection and do not affect the specific biochemical activity of discussed polypeptide.Also known Pegylation can increase the water solublity of discussed polypeptide, thus improves its preparation simplification.The example of numerous PEGylated polypeptides is known in the art and comprises: Mircera ^tM, the PEGylated forms of erythropoietin; Neulasta ^tM, the PEGylated forms of human granular leukocyte colony stimulating factor; Pegasys ^tMthe PEGylated forms of human interferon-alpha-2a; Peg-intron ^tM, the PEGylated forms of human interferon alpha-2 b; With glycol interferon λ 1 (at present just in clinical development).Therefore, the general exploitativeness disclosing the general compatibility that any PEGylated polypeptides and ON chelate are provided of the compatibility of a kind of PEGylated polypeptides (i.e. PEG ylated compound) in compositions and ON chelate, and particularly for the PEGylated forms (i.e. thymosin α1, Interferon Alpha-2b and interferon lambda 1) of the specific non-PEGylated polypeptides compatible with ON chelate of presentation in the disclosure.

Some antiviral drugs based on ON are being developed at present and are being used for the treatment of viral infection, and they comprise the phosphorothioate NAP REP 9AC (REP 2055), the REP 9AC ' (REP 2139) and REP 9AC that are used for the treatment of HBV ^m(REP 2148); Be used for the treatment of the miravirsen (Janssen etc., 2013, NEJM, March 27) of HCV; With the ALN-RSV01 (Zamora etc., 2011, Am.J.Resp.Crit.Care Med., 183:531-538) being used for the treatment of respiratory syncytial virus (RSV).The mechanism of action that they are had nothing in common with each other: as described in EXAMPLE V, NAP stops HbsAg to discharge into blood (a kind of protein of Immunosuppression function); Miravirsen (a kind of miRNA) blocks the effect of micro rna mir-122, and known RNA mir-122 plays a role in HCV copies; Block the synthesis of RSVN nucleocapsid protein with ALN-RSV01 (a kind of siRNA), thus stop RSV virion to produce.All these ON medicines are very effective for causing its Expected Results in experimenter: REP9AC/REP 9AC'/REP 9ACm blocks intracellular transport and the secretion of HBV subviral particle (SVP), this causes the removing of HBsAg in blood, and then causes the minimizing of HBV virus in blood; Mir-122 function involved by during miravirsen copies suppression HCV is very effective; And ALN-RSV-01 produces very effective to blocking-up RSV capsid protein.But, at these compounds based on ON of parenteral administration in any case, when by intravenous infusion, they with such as generate heat, shiver with cold, to shake etc. that it is relevant to use related side effects, or when being used by subcutaneous administration with the pain of injection site, inflammation or harden relevant.The more important thing is, although all these medicines based on ON have its Expected Results in experimenter, overall therapeutic outcome from hope result also away from: only have sub-fraction with the patient that these medicines carry out treating realize substantive antiviral response or treatments period or stop treatment after control completely its infect.

Therefore wish these antiviral drugs based on ON any are prepared as ON chelate (reduce to minimum to make it use related side effects and improve its pharmacokinetics potentially) and they and one or more antiviral polypeptides (as PEG ylated compound, Interferon Alpha-2b or thymosin α1 or glycol interferon λ 1) are combined in same preparation.Join together to be to cause in experimenter the beneficial effect that the antiviral response aspect more comprehensively strengthened has raising based on the specific antivirus action of compound (in chelate) of ON and the immunostimulation antivirus action of polypeptide, and compared with being used alone with any one medicine, make most subjects realize the control completely of its infection.

It may be useful for infecting with the antiviral response realizing improving in experimenter with the medicine composite for curing specific virus comprising at least one antiviral ON chelate and at least one antiviral polypeptide or PEGylated polypeptides.

It may be useful for infecting with at least one antiviral ON chelate and antiviral polypeptide treatment specific virus, and wherein often kind of these compound is used separately in different pharmaceutical compositions, and no matter whether route of administration is identical.

In order to provide antiviral response as well as possible in experimenter, be necessary to add the third non-ON, non-polypeptide medicine in antiviral ON chelate and the combined therapy of antiviral polypeptide (no matter whether using with identical or independent pharmaceutical composition).These medicines can be (but being not limited to): Entecavir, tenofovir disoproxil fumarate, telbuvidine, adefovir ester, lamivudine, ribavirin, VX-960, Bo Saipuwei, GS-7977, tegobuvir, zanamivir, Oseltamivir, ganciclovir, FOSCARNET, acyclovir, azidothymidine AZT, Abacavir, Lopinavir, ritonavir and/or efavirenz.These antiviral drugs can stop the viral genome of much virus and or the copying, as HCV, HBV, HIV, influenza virus, RSV and cytomegalovirus of virus mRNA.

Likely specific antivirus action and the antiviral polypeptide of antiviral ON chelate or the immunostimulation of PEGylated polypeptides are combined and can be provided best in the experimenter suffering from viral infection by the combination of non-ON listed above, non-polypeptide drugs block viral DNA/rna replicon and the most effectively treat and respond and increase the chance that the experimenter discussed obtains the lasting control of virus further, and this control is lasting after can breaking in the treatment.

ON chelate in preparation can derive from any ON with antiviral activity, and the example provides in Table 1.

table 1

The example of the ON of chelate can be prepared into.

LNA=locks nucleic acid, PS=thiophosphate, 2'OMe=2 ' O methyl, 5'MeC=5 ' methylcystein

Polypeptide in preparation can have direct antiviral activity or can stimulation of host source antiviral activity and can be following:

Thymosin α1;

Any alpha-interferon or its polyethylene glycol derivative;

Any beta-interferon or its polyethylene glycol derivative;

Any gamma interferon or its polyethylene glycol derivative;

Any λ-interferon or its polyethylene glycol derivative;

Intederon Alpha-2a or α-2b or α-N3;

Interferon beta-1a or β-1b;

Gamma interferon 1-b;

Interferon lambda 1 or λ 2 or λ 3;

PEG ylated compound or α-2b or λ 1 or λ 2 or λ 3;

Myrcludex B；

Any antiviral cell factor or its polyethylene glycol derivative;

Thymus protein A; And/or

Any display has polypeptide or the PEGylated polypeptides of antiviral activity or immunostimulatory activity.

There is provided a kind of demonstration herein, namely numerous different ON chelate can with homopolypeptide or PEGylated polypeptides drug regimen be in single medicine compositions, and this does not affect the structure of ON or polypeptide.

These compositionss may be used for needing the experimenter of this treatment simultaneously and adopting single administration mode to use ON chelate and polypeptide drugs, as in following examples VI prove).

In addition, above compositions can comprise physiology and/or pharmaceutically acceptable supporting agent, adjuvant, vehicle and/or excipient.The feature of supporting agent may depend on route of administration.Term " pharmaceutically acceptable supporting agent, adjuvant, vehicle and/or excipient " refers to and can use experimenter, to be incorporated in the present composition and not to destroy the supporting agent of its pharmacological activity, adjuvant, vehicle or excipient.Can be used for the pharmaceutically acceptable supporting agent of pharmaceutical composition described herein, adjuvant, vehicle and excipient include but not limited to following: ion-exchanger, aluminium oxide, aluminium stearate, lecithin, self-emulsifying drug delivery systems (" SEDDS "), the surfactant used in pharmaceutical dosage form is as Tweens or other similar polymeric delivery matrices, serum proteins are as human serum albumin, buffer substance is as phosphate, glycine, sorbic acid, potassium sorbate, the partial glyceride mixture of saturated vegetable fatty acid, water, salt or electrolyte are as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, silica sol, magnesium trisilicate, polyvinylpyrrolidone, based on cellulosic material, Polyethylene Glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, Polyethylene Glycol, lanoline, Capric acid sodium salt or TDM (TDM), or other TDM derivant of alkylation sugar.Cyclodextrin as α-, β-and gamma-cyclodextrin, or the derivant of chemical modification is as hydroxyalkyl cyclodextrin, comprises 2-and 3-HP-β-CD, or other derivant of dissolving also may be used for strengthening sending of the present composition.

Compositions described herein can comprise other treatment agent as described below, and can such as prepare by using the medicated premix (such as, excipient, binding agent, antiseptic, stabilizing agent, flavoring agent etc.) of traditional solid or liquid vehicle or diluent and the type that is suitable for mode to be applied according to such as those technology of knowing in field of pharmaceutical preparations.

Compositions described herein can be used by any suitable method, such as, Orally administered, such as with tablet, capsule, granule or powder form; Sublingual; Oral cavity; Parenteral, such as by subcutaneous, intravenous, intramuscular, in sheath or injection or infusion techniques (such as, as sterile injectable aqueous or non-aqueous solution or suspension); Suck; Locally, as with emulsifiable paste or ointment; Or rectum, as with suppository or enema forms; To comprise the dosage unit preparations of pharmaceutically acceptable nontoxic vehicle or diluent.This compositions can such as be used with the form being suitable for instant-free or delay to discharge.Instant-free or delay release can by using suitable pharmaceutical composition to realize, or, particularly when delaying release, realize by using the such as device such as hypodermic implant or osmotic pumps.Therefore, above compositions may be applicable to being used by any one in following approach: ophthalmic, oral, Sublingual, intestinal, suction, subcutaneous injection, intramuscular injection, peritoneal injection, intrathecal injection or infusion, tracheal strips, intravenous injection or infusion, or local.

Suspension and immediate release tablet is comprised for Orally administered exemplary composition, described suspension may comprise such as giving the microcrystalline Cellulose of globality, as alginic acid or the sodium alginate of suspending agent, as the methylcellulose of viscosity intensifier, with sweeting agent or flavoring agent, such as those known in the art; And described immediate release tablet can comprise such as microcrystalline Cellulose, starch, magnesium stearate and/or lactose and/or other excipient, agent, disintegrating agent, diluent and lubricant are amassed in binding agent, increasing, such as those known in the art, they do not disturb oligonucleotide chelate stability.This compositions also can be come via oral delivery by Sublingual and/or oral administration.Molded tablet, compressed tablets or lyophilizing tablet are operable exemplary form.Exemplary compositions comprises prepares those of this compositions with the fast dissolving diluents such as such as mannitol, lactose, sucrose and/or cyclodextrin.The such as high molecular weight excipients such as cellulose (avicel) or Polyethylene Glycol (PEG) can also be comprised in these preparations.These preparations can also comprise excipient to help mucosal adhesive, as hydroxypropyl cellulose (HPC), hydroxypropyl emthylcellulose (HPMC), sodium carboxymethyl cellulose (SCMC), copolymer-maleic anhydride (such as, and the medicament (such as, Carbopol 934) of the Co ntrolled release such as such as acrylic copolymer Gantrez).For ease of manufacturing and using, lubricant, fluidizer, flavoring agent, coloring agent and stabilizing agent can also be added.

The effective dose of compound described herein can be determined by those skilled in the art, and comprise exemplary dosage, per kilogram of body weight every day about 0.1 to 50mg reactive compound for adult, this can use with single dose or with the form of the dosage independently separated, as (but being not limited to) every day 1 to 5 time, or 1 to 7 dosage weekly.Be understood that, for any specific experimenter, specific dosage level and administration frequency may be different, and will many factors be depended on, comprise the order of severity of the activity of specific compound used, the metabolic stability of that compound and effect duration, the species of experimenter, age, body weight, general health, sex and diet, method of application and time, excretion rate and clearance rate, drug regimen and very pathology.The preferred experimenter for the treatment of comprises animal, be most preferably mammalian species as people, and domestic animal is as dog, cat etc.

Also provide herein when combinationally using so that the example of the useful antiviral activity of antiviral oligonucleotides chelate and antiviral polypeptide when treating the people patient suffering from viral infection.

More easily the disclosure will be understood by reference to following examples.

example I

The sign of degeneracy ON

Figure 1A describes in detail by co-injection while of HPLC (use drainage column) separation to kind of the ON preparation of two in pillar.Wherein first is called internal standard substance and is the 21 mer phosphorothioate esterification ON with specific defined nucleotide sequence, and second is REP 2006 (40 aggressiveness degeneracy phosphorothioate ON).These two kinds of materials are only separated into different definition peaks according to its physicochemical property (i.e. size and hydrophobicity); The nucleotide sequence existed in these ON each does not have material impact to its physicochemical property and is therefore separated not impact to it.Therefore, internal standard substance flows out pillar as the peak of the strict difinition with the less retention time compared with REP 2006, just varying in size due to these two kinds of ON polymer.Notice that at the acromion of any side, REP 2006 peak be due to the failure sequences existed usual in the preparation of longer ON.Although the sequence character heterogeneity of REP2006, but it resolves to similarly well-defined peak by HPLC as 21 aggressiveness particular sequences, this illustrates the common physicochemical property of all substances in REP 2006 preparation, even if there are the different sequences of squillion.After being separated REP 2006 and 21 aggressiveness peak by HPLC, they can carry out mass spectrum (MS) and analyze the material (Figure 1B and 1C) existed in these definition peaks with qualification.

In fig. ib, 21 aggressiveness are resolved into the one matter of the MW with 7402.6Da, consistent with this PS-ON of the sequence with definition.But the MS of REP 2006 analyzes (Fig. 1 C) display and there is maximal number desired substance, and their mass range has almost ideal normal distribution, consistent with the character of its complete degeneracy.This mass range is from C40 (minimum material) to A40 (maximum material) and the occurrence rate of these materials (prevalence) is minimum, along with these material masses are close to the center of mass range, the number of material raises (peak intensity).This is because the different sequences of increasing number will cause similar quality.The all different ON materials existed in REP 2006 have this fact of identical retention time between HPLC separation period on drainage column to be proved to have formed objects and the physicochemical property may with all ON of identical chemical modification (i.e. phosphorothioate) with highly similar (if not identical) clearly, and therefore, can think functional similarity in any application not relying on the nucleotide sequence be present in specific ON molecule or character.Therefore, form the ON sequence that can not depend on existence with any ON chelate that any specific degeneracy ON (such as REP2006) is observed, and the conservative physicochemical property of any ON must be depended on.

example II

Chelate is formed with antiviral ON

REP 2031, REP 2055, REP 2057 and REP 2139 are nucleic acid polymers (the NAP) (Bernstein etc. of the broad anti-viral activity had for HIV, HSV, cytomegalovirus, LCMV, HCV and other enveloped viruses, 2008, Antimicrobial Agents Chemother.52:2727-2733; Cardin etc., 2009, Virology are J.6:214; Vaillant etc., 2006, Antimicrobial Agents Chemother., 50:1393-1401; Guzman etc., 2007, Antiviral Therapy, 12:1147-1156; Lee etc., Virology, 372:107-117; Matsumura etc., 2009, Gastroenterology 137:673-681).All these compounds are all the ON of the complete phosphorothioate of 40 aggressiveness, when REP 2031 (SEQ ID NO:1) with 5 '-3 ' sequence C ₄₀, when REP 2055 (SEQ ID NO:2) with (AC) ₂₀, when REP 2057 (SEQ ID NO:3) with (AG) ₂₀and when REP 2139 (SEQ ID NO:18) with (2'OMeA-2'OMe, 5'MeC) ₂₀.Although REP 2031, REP 2055 and REP 2057 are DNA ON, REP 2139 is RNA ON, wherein all ribose all to be modified and all cytosine all methylates through 5 ' through 2 ' O methyl.

Forming chelate with these antiviral ON is use the 3'FITC labeled derivative thing fluorescence polarization of these compounds to study.Between ON synthesis stage, the reagent of good foundation and synthetic schemes is used to be conjugated on Fluorescein isothiocyanate (FITC) by each ON by 3 carbon joints of rigidity at 3 ' end.These ON cracking and staying as ammonium salt from synthesis.These ON are prepared to the 0.5mM storing solution (pH 7.2) in 1mM TRIS separately.These storing solutions are used at FP buffer (10mM TRIS, 80mM NaCl, 1mM EDTA, 10mM beta-mercaptoethanol and 0.1% -20) preparation 3nM fluorescence ON solution in.The EDTA existed is for removing any divalent metal existed in solution before FP measures.Often kind of these buffer solution also comprises 80mM NaCl and is formed with the ON complex evaluated under the univalent cation of molar excess exists.ACS level bivalence (2+) metal chloride salt (as described in table 2) is added to often kind of fluorescence ON in solution.The formation of dimer or high-order ON chelate is monitored by the rising (quantitative by dimensionless unit " mP ") of fluorescence polarization, and therefore ON chelate formation increase causes quality to have larger change (see Fig. 3).These ON chelate rollings in the solution caused are slowed down and are caused the polarization of emitting fluorescence to increase (see Fig. 4).The result of these experiments provides in table 2.

table 2

ON chelate is formed with different divalent metal and NAP

Meansigma methods and standard deviation are based on twice repeated measure.

Under various condition, all fluorescently-labeled ON observe the remarkable increase of fluorescence polarization under calcium and magnesium exist, and show to form ON chelate with these divalent metals.These results prove to draw a conclusion:

REP 2006, REP 2031, REP 2055, REP 2057 and REP 2139 form dimer and high-order complex under magnesium and calcium cation exist.Estimate all to form these complex (as shown in degeneracy NAP REP 2006) with every other multivalent metal cation and with any ON.The formation of these ON complex relates to the interaction of these ON and these divalent metals.

The formation of ON complex can not be due between nitrogenous base by traditional interactional hybridization of Wo Sen-Ke Like because REP 2031, REP 2055, REP 2057 and REP2139 can not self hybridization under the experiment condition used.

The formation of these ON complex is stable and water soluble solution, and because these complex seem to be incorporated with the part of discussed divalent metal as the complex formed, these ON complex have the effect of the divalent metal discussed from chelating the solution forming described ON complex.

eXAMPLE III

Comprise the preparation of the compositions of ON chelate and polypeptide.

The preparation comprising the compositions of ON chelate and polypeptide is that different antiviral ON and four kind of the antiviral polypeptide of use five kinds carries out.The ON used previously had been presented in enveloped virus the NAPS with broad anti-viral activity, and these enveloped viruses comprise HBV, HCV, influenza virus, RSV, Ebola (Ebola) virus, HSV-1 and HSV-2.These ON are REP 2006 (40 aggressiveness degeneracy phosphorothioate NAP), REP 2055 (SEQ ID NO:2), REP 2057 (SEQ ID NO:3), REP 2139 (SEQ ID NO:18) and REP2148 (SEQ ID NO:11).These NAP be in movable reactor by the solid phase synthesis condition of standard prepare and during purification, carry out in addition salt exchange so that ammonium relative ion is replaced as sodium relative ion.The theoretical free acid molecule amount of these ON is (12612-13092), 12612,13413 and 14094 and 12893Da respectively.The antiviral polypeptide used is Interferon Alpha-2b, thymosin α1, PEG ylated compound and interferon lambda 1 (IL-29), their whole displays have antiviral activity (Yang etc., 2008, Antiviral Res., 77:136-141; Fried etc., 2002, N.Engl.J.Med., 347:975-982; Friborg etc., 2013, Antimicrob.Agents Chemother.57:1312-1322).All ON are the working standard synthesis of synthesizing for solid phase oligonucleotide according to this area, and use the method for generally acknowledging to be prepared into the sodium salt being suitable for using in body, and before storage lyophilizing to water content < 10%.REP 2006, REP 2055, REP 2057 and REP 2148 are the non-pure preparations containing a small amount of not exclusively synthetic product usually occurred during solid phase synthesis.REP 2139 is prepared to the preparation of the higher degree that there is not the incomplete synthetic product of major part.REP2006, REP 2057 and REP 2139 is prepared into solution in advance and is adjusted to 25mg/ml between chelate Formation period in normal saline.REP 2055 is also prepared into solution in advance in normal saline, but is adjusted to 12.5mg/ml during chelate preparation.Thymosin α1 has 28 aminoacid and molecular weight is the synthetic peptide of the amino terminal acidylate of 3108Da, and can commercial formulation (Zadaxin ^tM) obtain, and in water for injection, be prepared into 1.6mg/ml solution.Interferon Alpha-2b is prepared by recombinant DNA technology in escherichia coli (E.coli), and has the molecular weight of 19271Da.Interferon Alpha-2b is with commercial formulation (IntronA ^tM) obtain, and 1X10 is prepared in the water for injection containing 1mg/ml human serum albumin (there is the molecular weight of about 66500Da) ⁷the solution of IU/ml.PEG ylated compound is the covalent conjugates of Intederon Alpha-2a and single branch double focusing glycol monoethyl ether chain, and its total approximate molecular weight is 60000Da.PEG ylated compound is with commercial formulation (Pegasys ^tM) obtain, its pre-dilution in the water for injection containing trace benzylalcohol becomes the concentration of 360ug/ml.The recombinant interferon λ 1 (IL-29) of purification is from Ebiosciences (SanDiego, U.S.A.) obtain as the 0.5mg/ml no-load agent solution in phosphate buffered saline (PBS), and as gel electrophoresis measure, it has the molecular weight of about 20kDa.

Calcium chloride is as CaCl ₂2H ₂the 100mg/ml USP preparation (Lifeshield of O in water for injection ^tM, Hospira) obtain.Magnesium sulfate is as MgSO ₂7H ₂that the 500mg/ml USP preparation (Baxter) of O in water for injection obtains and be diluted to 100mg/ml in normal saline.In order to prepare the ON chelate containing calcium, magnesium or calcium and magnesium, these metal salt solutions are dropwise added in ON solution gradually, until reach the slaine of hope and the ratio of ON under constantly mixing.For calcium chelate, obtain the ratio of every 100mg ON 30mg calcium chloride in the solution, except REP 2148 calcium chelate, its every 100mg ON contains 20mg calcium chloride.For magnesium chelate, obtain the ratio of every 100mg ON 30mg magnesium sulfate in the solution.For calcium mixture/magnesium chelate, obtain the ratio of every 100mg ON 15mg calcium chloride and 15mg magnesium sulfate in the solution.Verified this method causes the formation (as described in U.S. Application Publication number 2012/0046348) of ON chelate.When this EP (end of program), ON chelate has very light lurid clear homogeneous solution.

ON chelate/polypeptide or PEGylated polypeptides compositions are prepared to 1ml final volume with polypeptide or PEGylated polypeptides solution by the ON chelate solution of light and slow mixing 1: 1 ratio.

eXAMPLE IV

Comprise the sign of the compositions of ON chelate and polypeptide.

In order to confirm the concordance of ON and polypeptide or the PEGylated polypeptides comprised in the compositions prepared in EXAMPLE III, high pressure lipuid chromatography (HPLC) (HPLC) analysis is carried out to them, carries out electrospray ionization mass spectrometry (ESI-MS) analysis subsequently.Because quality and the chemical property of the ON existed in these compositionss and polypeptide or PEGylated polypeptides material have relatively large deviation, so use three kinds of different HPLC methods to analyze:

Oligomer method: solid phase: 2x 50mmACE ^tMc18 (3um)

Mobile phase: A=1/0.1%HFIPA/DIEA

B＝65/0.075/0.0375％ACN/HFIPA/DIEA

Gradient: 5-25%B in 20min

70%B maintains 2min, 60 DEG C

Flow velocity: 0.4ml/min

Method of protein 1: solid phase: 2x 50mm PLRP-s (Agilent ^tM) 4000A (8um)

Mobile phase: A=is containing the ACN of 0.05%TFA

B=is containing the ACN of 0.05%TFA

C=is containing 0.1%NH ₄the H of OH ₂o

D=is containing 0.1%NH ₄the 40/40/20ACN/MeOH/H of OH ₂o

Gradient: 80/20%C/D washs 1min to waste liquid

(optional 20%D washs 1min to waste liquid)

In 15min, 20%-100%D, 100%B maintain 2min

(in optional 16min 20-70%B)

Flow velocity: wash as 0.5ml/min

Gradient is 0.3ml/min

Method of protein 2: solid phase: 2x 50mm PLRP-s (Agilent ^tM) 4000A (8um)

Mobile phase: A=is containing the ACN of 0.05%TFA

B=is containing the ACN of 0.05%TFA

C=is containing 0.1%NH ₄the H of OH ₂o

D=is containing 0.1%NH ₄the 40/40/20ACN/MeOH/H of OH ₂o

Gradient: 80/20%C/D washs 1min to waste liquid

80/20%A/B-100%B in 15min

100%B maintains 2min

Flow velocity: wash as 0.5ml/nin

Gradient is 0.3ml/min

The instrument used is LTQ-Orbitrap Discovery, for oligomer method, is run with LTQ scan pattern with anion, and for two kinds of method of proteins, is run under 7500 resolution with Orbitrap fine scanning pattern with cation.Synonym: ACN=acetonitrile, TFA=trifluoroacetic acid, HFIPA=hexafluoroisopropanol, DIEA=N, N-diisopropylethylamine.

The result analyzed from these is found in Fig. 5-95.All HPLC chromatograms are mapped according to total ion current (TIC) data, wherein peak retention time is with the quality identification (except Figure 39,43,47,51,55,77,81,91 and 95, wherein this is Polyethylene Glycol conjugate because existing in PEG ylated compound and is prevented from) from ESI-MS subsequently.For the case of the compositions containing ON chelate and thymosin α1, described oligomer method is enough to analyze two kinds of materials simultaneously.For other compositionss containing macromolecular weight protein, ON analysis and polypeptide analysis are carried out to same sample, but use different HPLC method (as shown in Fig. 5-95).

The LC-MS analytical proof ON be present in these compositionss of the ON content of all compositionss meets its expection purity: for REP 2055 (Fig. 5,24,40,56,68,71,74 and 78), REP 2057 (Fig. 9,27 and 44) and REP 2148 (Figure 17,33,52 and 64), in using the HPLC of oligomer method to analyze, observe a small amount of not exclusively ON synthetic product, but do not exist in REP 2139 (Figure 13,30,48,60,82,85,88 and 92).Under any circumstance, the ESI-MS leading (elementary) ON peak is almost completely relevant to the expection molecular weight of REP2055, REP 2057, REP 2139 and REP 2148.For REP 2006, the ESI-MS of main peak produces the peak of wider distribution in 12612-13092Da scope, consistent with its degeneracy character (Figure 22 and 37).

For the analysis of the compositions containing Interferon Alpha-2b, the HPLC chromatogram display large low resolution peak (Fig. 7,11,15 and 19) consistent with there are more than one polypeptide in these compositionss using method of protein 1 to obtain, but main peak resolves to the material (Fig. 8,12,16 and 20) of two kinds of almost almost completely relevant to the expection molecular weight of Interferon Alpha-2b and human albumin different qualities.

For the compositions containing thymosin α1, the HPLC chromatogram obtained from oligomer method 1 can obtain second main peak same first separation, but because the molecular weight of this peptide species is relatively low, its retention time ratio (elementary) ON peak little many (Figure 21,24,27,30,33,68,71,82 and 85).In all cases, the expection molecular weight of ESI-MS and the thymosin α1 of this secondary peak almost completely relevant (Figure 23,26,29,32,35,70,73,84 and 87).

For the compositions containing PEG ylated compound, the existence of PEGylated conjugate makes LC-MS analysis greatly complicated, and PEGylated conjugate produces numerous ionic species, and this makes the mass spectrographic correct inverting of any PEGylated polypeptides be difficult to.But, use method of protein 2, the HPLC data from the compositions containing PEG ylated compound all show the similar main peak (Figure 38,42,46,50,54,76,80,90 and 94) consistent with the retention time of expecting for PEGylated polypeptides.The ESI-MS at these peaks analyzes the similar group (Figure 39,43,47,51,55,77,81,91 and 95) of the Pegylation coherent signal shown from m/z600-100-with 1500-3600 consistent with the existence of pegylated protein in all cases.

A part in the whole oligonucleotide chelate-peptide compositions prepared in EXAMPLE III has also carried out calcium and or magnesium determination by inductively coupled plasma emission spectroscopy (ICP-OES).Calcium and the magnesium determination data of often kind of compositions analyzing provide in table 3.

table 3

The metal assay of ON chelate-peptide composition.

Below the result proof of EXAMPLE III and IV:

1. the compositions comprised containing calcium, magnesium or the calcium/magnesium of mixing and the ON chelate of polypeptide or PEGylated polypeptides can be prepared into and be suitable for parenteral administration.

2. as by between the actual measurement of REP 2055, REP 2057, REP 2139, REP 2148, Interferon Alpha-2b, thymosin α1 and interferon lambda 1 and theoretical molecular almost completely dependency prove, the ON in these compositionss discussed and polypeptide or PEGylated polypeptides all do not experience any chemical change detected.

3. when comprising the compositions of PEG ylated compound, ON does not change, and polypeptide change (if there is) can not detect.

According to these effects, following deduction can also be had:

1.REP 2006 is the ON of complete degeneracy and is therefore the prototype model of the common physicochemical property of all ON.Therefore, any ON that can be formulated as chelate in the solution will with these polypeptide or PEGylated polypeptides compatible.

2.REP 2006, REP 2055, REP 2057 and REP 2148 are DNA, and REP2139 is RNA.Therefore, any RNA or DNA or RNA/DNA can be used in the preparation of compositions comprising ON chelate and polypeptide to hybridize ON.

3.REP 2006, REP 2055 and REP 2057 have the ribose of unmodified, and each ribose of REP2139 is methylated by 2 ' O.Therefore, any 2 ' ribose is modified all by compatible with the preparation of the compositions comprising ON chelate and polypeptide.

Each cytosine that 4.REP 2139 and REP 2148 comprises is modified to 5 ' methylcystein further.Therefore, the ON comprising modified base may be used for preparing the compositions comprising ON chelate and polypeptide or PEGylated polypeptides.

5. the compositions containing ON chelate and polypeptide or PEGylated polypeptides will be compatible with any pharmaceutically acceptable divalent metal such as such as calcium and/or magnesium etc.

6. four peptide species used in compositions preparation contain the structure differed greatly: thymosin α1 is a kind of little improvement on synthesis, Interferon Alpha-2b and the interferon lambda 1 large recombinant peptide that to be large recombinant polypeptide and PEG ylated compound be with large complicated PEGylated conjugate.Therefore, can use from little improvement on synthesis to complicated conjugate as the large recombinant polypeptide of Polyethylene Glycol these widely polypeptide successfully prepare the compositions containing ON chelate and polypeptide.Likely unique restriction is exactly discussed polypeptide should water soluble solution.In addition, in intron A (source as Interferon Alpha-2b), BSA is used not affect the stability of the compositions prepared with this polypeptide formulations as carrier protein.

7. described above, Pegylation well known in the art can be used for improving the toleration of polypeptide and making a mistake property of pharmacokinetics and numerous PEGylated polypeptides uses (see above) as approval medicine at present.Therefore, confirm that compositions described herein can tolerate the existence of Pegylation peptide and combination ON chelate and Pegylation peptide well for improving antiviral response and the practicality in people patient (see following examples VI) instruction clear and definite for any person skilled in the art provides, any polypeptide namely imagined herein as a part for compositions can also exist as PEGylated polypeptides.Such as, thymosin α1 can by Pegylation, and Interferon Alpha-2b can by Pegylation (the PEGylated forms Peg-intron of Interferon Alpha-2b ^tMthe medicine of approval at present), and interferon lambda 1 can by Pegylation (PEGylated forms of interferon lambda 1 be at present clinical middle as antiviral agent exploitation).

eXAMPLE V

NAP suppresses HBsAg to transport out cell.

Prove that hbs antigen (HBsAg) blocks many aspects (cheng etc., the J.Hepatology 43:465-471 to the immunne response of HBV infection; Moucari etc., Hepatology 49:1151-1157; Vanlandschoot etc., J.Gen.Virol.83:1281-1289; Woltman etc., PloS One 6:e15324; Wu etc., Hepatology 49:1132-1140 and Xu etc., Mol.Immunology 46:2640-2646).Therefore, eliminating circulation HBsAg may be the key factor making to suffer from the recovery of chronic hepatitis-B infection patient immunocompetence.A kind of effective ways eliminating the HBsAg in circulation prevent SVP from being formed and or discharge (SVP is the main carriers of HBsAg blood) from infection cell.The form of SVP occurs and intracellular transport can the little form (sHBsAg) by HBsAg expression albumen in BHK-21 cell be carried out in-vitro simulated, and sHBsAg is the form of special enrichment in SVP.The alternative model (Patient etc., J.Virology 81:3842-3851) that the form that this model system is considered to the HBV SVP in people patient occurs and transports.Because serum HBsAg has pivotal role in the chronicity allowing HBV infection, SVP therefore in this model of compounds block is formed or the ability of its intracellular transport shows its antiviral activity to HBV.

Multiple NAP compound is tested in the BHK-21 cell of expressing sHBsAg, comprise phosphorothioate NAP REP 2006 and the REP 2107 (all ribose of 2107 are also modified with 2 ' O methyl) of complete degeneracy, non-phosphorothioate, complete 2 ' O methylated degeneracy NAP (REP 2086) and the NAP:REP 2055 (SEQ ID NO:2) be made up of poly-AC sequence and REP 2148 (SEQ ID NO:11).These NAP introduce BHK-21 cell by electroporation with the template ribonucleic acid using electroporation to express for sHBsAg simultaneously.By observing to the position of HBsAg albumen in BHK-21 cell the activity evaluated in BHK model system by immunofluorescence microscopy.If HBsAg is confined to perinuclear space and stops transporte to cells periphery (secretion) just to judge that compound is that tool is activated.The Activity Summary of various NAP compound is in following table 4.

table 4

Multiple NAP is on the impact of the HBsAg transhipment in BHK-21 cell

NAP	Be retained in the HBsAg in perinuclear space	The HBsAg of transporte to cells periphery
			Contrast (there is not NAP)	-	++++
REP2006	++++	-
			REP2107	+++	+
REP 2086	-	++++
			REP 2055(SEQ ID NO：2)	++++	-
REP 2148(SEQ ID NO：11)	++++	-

Not-=do not observe effect

+ to +++ +=observe critical to effect completely

The result processing the BHK-21 cell of expressing sHBsAg with REP 2006 and REP 2107 illustrates that NAP can block sHBsAg transhipment in the mode that sequence is irrelevant.REP 2086 lacks the active existence that this activity strictly depends on phosphorothioate that illustrates.In addition, under 2 ' ribose modifies (in REP 2107) and base modification (being 5 ' methylcystein REP 2148) existence, this ability is retained.In addition, known REP 2107, REP 2055 and REP 2139 are completely with REP 2006 quite active without any immunostimulatory activity.Equally, the defined nucleotide sequence (REP 2055 and REP 2148) of poly-AC is suitable with the activity of degenerate sequence (REP 2006 and REP 2107).

These results show, degenerate sequence and comprise AC repeat (and be therefore also CA repeat) sequence and also repeat (as TG and GT or UG and GU) containing alternate purine/pyrimidine nucleotide and modify containing 2 ' ribose or base modification or other sequences simultaneously containing 2 ' ribose modification and base modification (REP 2139 see in example VI) situation in, under the oligonucleotide length of 20-120 nucleotide, the NAP of expection phosphorothioate can block SVP formation and from the intracellular transport of infection cell and secretion, as United States Patent (USP) 8, 008, 269, 8, 008, 270 and 8, 067, described in 385.

example VI

Be used for the treatment of the combined therapy of the chronic hepatitis B of people patient

REP 2055 is simple sodium salts of phosphorothioate ON (SED ID NO:2).REP2139-Ca uses often to there is 100mg oligonucleotide and just have 30mg CaCl in normal saline ₂the calcium chelate of phosphorothioate oligonucleotide REP 2139 (SEQ ID NO:18) prepared of ratio.REP 2055 and REP 2139 is NAP and this compounds is to the effective broad-spectrum disease resistance cytotoxic compound of enveloped virus (Bernstein etc., 2008, Antimicrobial Agents and Chemotherapy, 52:2727-2733; Cardin etc., 2009, Virology Journal, 6:214; Guzman etc., 2007, Antiviral Therapy, 12:1147-1156; Lee etc., 2008, Virology, 372:107-117; Matsumura etc., 2009, Gastroenterology, 137:673-681; Vaillant etc., 2006, Antimicrobial Agents and Chemotherapy, 50:1393-1401 and United States Patent (USP) 8,008,269B, 8,008,270 and 8,067,385).Chelate does not affect the biological activity of NAP or other ON materials and therefore REP 2139 is formulated as calcium chelate and does not affect its antiviral activity.The NAP modification that antiviral activity uniquely needs is the phosphorothioate of each key in ON.Other modification comprising 2 ' ribose modification (as 2 ' O methylates) and base modification (as 5 ' methylcystein and/or 4 ' paper substrate) can be ignored the impact of the antiviral activity of NAP, but may be used for the toleration optimizing people patient.

PEG ylated compound by Roche Inc. (Basel, Switzerland) with trade mark Pegasys ^tMsell and ratify to be used for the treatment of chronic HBV infection.Thymosin α1 by SciClone Pharmaceuticals (Foster City, California, U.S.A.) with trade mark Zadaxin ^tMsell and be also used for the treatment of chronic HBV infection in the approval of many Asian countries.

Whether can improve the antiviral response of the patient suffering from chronic HBV infection to study NAP treatment and the antiviral effect of the combination of immunization therapy (stimulating adaptability and born immunne response), patient carries out single therapy with REP 2055 and with REP 2139-Ca and thymosin α1 (Zadaxin ^tM-1.6mg subcutaneous injection, gives for twice weekly) or PEG ylated compound (Pegasys ^tM-180 μ g subcutaneous injections, give once in a week) carry out combined therapy, reach its ongoing NAP scheme.

REP 2055 and REP 2139-Ca has same isoreactivity when using in single therapy in the release blocking HBsAg, block the release of HBsAg be generally all NAP to the mechanisms of therapeutic action of HBV infection.REP 2055 and REP 2139-Ca when giving in suitable monotherapy regimen, the HBsAg realized in 9 in 7 respectively in 8 patients and in 12 patients in HBV infection patients serum removes, and the serum HBsAg of these two kinds of medicines is removed quite (see table 5), this display when the antiviral activity of these NAP as sodium salt or when giving as chelate suitable.

table 5

REP 2055 and REP 2139-Ca removes effectively and comparably the serum HBsAg suffering from the patient of chronic HBV infection.

* by Abbott Architect ^tMhBsAg detection by quantitative measures

Treatment based on interferon makes the patient of 25% realize control (serum HBV DNA < 500 copy/ml of its HBV infection usually after treatment stops; Mourcari etc., 2009, Hepatology 49:1151-1157), and it is generally acknowledged that thymosin α1 treatment has suitable effect (Yang etc., 2008Antiviral Research 77:136-141).

The control (serum HBV DNA < 500 copy/ml) of HBV infection is realized in 3 (43%) of REP 2055 single therapy after treatment stops in 7 patients.REP 2139-Ca treats, and (in removing serum HBsAg, having identical effect with REP 2055 when giving with single therapy) realizes virus control (see table 6) with thymosin α1 or being combined in 8 (89%) suffered from 9 patients of HBV infection of PEG ylated compound.

table 6

Lasting virological response (SVR) is realized with the NAP/ immunization therapy of NAP single therapy or combination.

* serum HBV DNA < 500 copy/ml

These results prove that the compound action of antiviral ON chelate (being REP 2139-Ca in this embodiment) and antiviral polypeptide (being PEG ylated compound or thymosin α1 in this embodiment) can make the treatment of people patient's HBV infection control afterwards to improve and prove the practicality with ON chelate and polypeptide or PEGylated polypeptides combined therapy in the patient suffering from viral infection further.

The compositions be made up of antiviral ON chelate and antiviral polypeptide will be desirable, because they can suffer from the total antiviral response (its practicality is open in the present embodiment) of the patient of viral infection (comprising HBV infection) by providing two kinds of medicaments to improve simultaneously.

Antiviral ON chelate in above-mentioned composition can comprise any antiviral ON as above and can particularly including REP 2055 (SEQ ID NO:3, REP 2139 (SEQ ID NO:18), REP 2148 (SEQ ID NO:11) or any other NAP compound blocking HBsAg transhipment described above when HBV.

Polypeptide in above-mentioned composition can comprise any antiviral polypeptide or Pegylation antiviral polypeptide and can particularly including Interferon Alpha-2b, glycol interferon alpha-2b, PEG ylated compound, thymosin α1, interferon lambda 1 or glycol interferon λ 1 when HBV.

Can recognize, combinations thereof therapy not only in HBV infection, and also has similar beneficial effect at other viral infection as hepatitis C virus, influenza virus, RSV and the effect that can resist viral ON or antiviral polypeptide or PEGylated polypeptides have in other viruses of response etc.Although use NAP in the present embodiment, other antiviral ON worked by sequence related mechanism being mixed with chelate when these improvement of people patient's antiviral result can use and combine with antiviral polypeptide or PEGylated polypeptides is realized.When using the correct combination of antiviral oligonucleotides chelate and antiviral polypeptide, these effects also can be effective more widely in other viral infection.Such as, NAP has extensive activity to other enveloped viruses many such as such as hepatitis C, influenza, respiratory syncytial virus and cytomegaloviruses, and therefore estimate being combined in antiviral response more much better than any one compound in single therapy for generation in infected subjects of NAP chelate and suitable antiviral polypeptide (can being thymosin α1 or PEG ylated compound, but also can being be more suitable for the another kind of antiviral polypeptide that specific virus infects).In another case, in order to improve the antiviral response suffering from hepatitis C infection patient, the chelate of miravirsen (SEQ ID NO:7) and interferon (Pegylation or non-Pegylation) can be combined.

Estimate also to occur in such as antisense or siRNA or miRNA with the beneficial effect that NAP chelate and thymosin α1 or PEG ylated compound prove or infect for specific virus on the ON chelate of other kind of class oligonucleotides such as the fit ON of exploitation or immunostimulatory oligonucleotide.Therefore, estimate that the method that the ON chelate and suitable antiviral polypeptide that derive from these ON kinds any combined will have therapeutic use showing to have in the various viral infection of response the antiviral oligonucleotides (as sodium salt or chelate) in single therapy or antiviral polypeptide or PEGylated polypeptides.

In view of above embodiment, it is contemplated that now that two or more antiviral ON chelate (such as comprise NAP and antisense ON or comprise NAP and antiviral siRNA) can combine with one or more antiviral polypeptides or PEGylated polypeptides (such as PEG ylated compound and thymosin α1 or thymosin α1 and glycol interferon λ 1), in same preparation or in multiple preparation, to be used by identical or different approach.

In the embodiment above, REP 2139-Ca is used by intravenous infusion, and PEG ylated compound or thymosin α1 are used by subcutaneous administration.Based on the instruction of above embodiment, those skilled in the art easily can predict that ON chelate and polypeptide can give and can predict to have identical beneficial effect with when using (by identical or different route of administration) any one medicament respectively by subcutaneous injection or intravenous infusion in same preparation (as at those preparations disclosed in above embodiment 3 and 4) now.

Beneficial effect in the antiviral environment that the combination treatment of ON chelate described herein and antiviral polypeptide or PEGylated polypeptides describes in this article predicts that using ON chelate and polypeptide or PEGylated polypeptides to have in single therapy at these two kinds of medicaments carries out combined therapy in a certain degree active Curing circumstance and have similar beneficial effect clearly.Such as, the antisense ON (therefore it has active anticancer) of involved in target on cancer gene can be formulated into ON chelate described herein and further with known normally use in single therapy time there is to a certain degree active anticancer immunotherapeutic agent combine.These Curing circumstance can comprise cancer, multiple sclerosis and Alzheimer.

Claims

1. a pharmaceutical composition, it comprises the ON chelate and at least one polypeptide that are made up of two or more intermolecular oligonucleotide (ON) connected by bivalent cation.

2. a pharmaceutical composition, it comprises the antiviral ON chelate and at least one antiviral polypeptide that are made up of two or more intermolecular antiviral ON connected by bivalent cation.

3. pharmaceutical composition as claimed in claim 1 or 2, wherein said polypeptide is by further Pegylation.

4. pharmaceutical composition as claimed any one in claims 1 to 3, wherein said polyvalent cation is the alkaline-earth metal with 2+ state of charge.

5. the pharmaceutical composition according to any one of Claims 1-4, wherein said divalent metal is the transition metal with 2+ state of charge.

6. the pharmaceutical composition according to any one of claim 1 to 5, wherein said divalent metal is the lanthanide series metal with 2+ state of charge.

7. the pharmaceutical composition according to any one of claim 1 to 6, wherein said divalent metal is the late transition metal with 2+ state of charge.

8. the pharmaceutical composition according to any one of claim 1 to 7, wherein said divalent metal is calcium.

9. the pharmaceutical composition according to any one of claim 1 to 7, wherein said divalent metal is magnesium.

10. the pharmaceutical composition according to any one of claim 1 to 7, wherein said divalent metal is ferrum (2+), manganese, copper or zinc.

11. pharmaceutical compositions according to any one of claim 1 to 7, wherein said bivalent cation is made up of two or more different divalent metal.

12. pharmaceutical compositions according to any one of claim 1 to 7, wherein said bivalent cation is made up of calcium and magnesium.

13. pharmaceutical compositions according to any one of claim 1 to 12, wherein said ON chelate comprises at least one double-strand ON.

14. pharmaceutical compositions according to any one of claim 1 to 13, wherein said ON chelate comprises the ON that at least one has at least one phosphorothioate bond.

15. the pharmaceutical composition according to any one of claim 1 to 14, wherein said ON chelate comprises the ON of at least one complete phosphorothioate.

16. pharmaceutical compositions according to any one of claim 1 to 15, wherein said ON chelate comprises the ON that at least one has the ribose that one 2 ' is modified.

17. pharmaceutical compositions according to any one of claim 1 to 16, wherein said ON chelate comprises at least one each ribose by the methylated ON of 2 ' O-.

18. pharmaceutical compositions according to any one of claim 1 to 17, wherein said ON chelate comprises at least one ON comprising at least one 5 ' methylcystein.

19. pharmaceutical compositions according to any one of claim 1 to 18, wherein said ON chelate comprise at least one wherein each cytosine be the ON of 5 ' methylcystein further.

20. pharmaceutical compositions according to any one of claim 1 to 19, wherein said ON chelate comprise at least one wherein each ribose to be methylated by 2 ' O-and wherein each cytosine is the ON of 5 ' methylcystein further.

21. pharmaceutical compositions according to any one of claim 1 to 12, wherein said ON chelate comprises the oligonucleotide being selected from SEQ ID NO:1-6 and 10-18.

22. pharmaceutical compositions as claimed any one in claims 1 to 3, wherein said ON chelate comprises the oligonucleotide being selected from SEQ ID NO:7-9.

23. pharmaceutical compositions according to any one of claim 1 to 22, wherein said polypeptide be following at least one:

Thymosin α1;

Any alpha-interferon or its polyethylene glycol derivative;

Any beta-interferon or its polyethylene glycol derivative;

Any gamma interferon or its polyethylene glycol derivative;

Any λ-interferon or its polyethylene glycol derivative;

Intederon Alpha-2a or α-2b or α-N3;

Interferon beta-1a or β-1b;

Gamma interferon 1-b;

Interferon lambda 1 or λ 2 or λ 3;

PEG ylated compound or α-2b or λ 1 or λ 2 or λ 3;

Myrcludex B；

Any antiviral cell factor or its polyethylene glycol derivative;

Thymus protein A; With

24. pharmaceutical compositions according to any one of claim 1 to 23, it is that preparation is for subcutaneous administration.

25. pharmaceutical compositions according to any one of claim 1 to 23, it is that preparation is for intravenous infusion.

26. pharmaceutical compositions according to any one of claim 1 to 23, its be preparation for the following route of administration of at least one: infusion in aerosol suctions, ophthalmic, oral, intestinal, intramuscular injection, peritoneal injection, intrathecal injection, sheath, tracheal strips, intravenous injection and locally.

27. pharmaceutical compositions according to any one of claim 1 to 26, wherein said ON chelate comprises at least one ON be made up of SEQ ID NO:2.

28. pharmaceutical compositions according to any one of claim 1 to 26, wherein said ON chelate comprises at least one ON be made up of SEQ ID NO:11.

29. pharmaceutical compositions according to any one of claim 1 to 26, wherein said ON chelate comprises at least one ON be made up of SEQ ID NO:18.

30. pharmaceutical compositions according to any one of claim 1 to 29, it comprises one or more following medicines further: Entecavir, tenofovir disoproxil fumarate, telbuvidine, adefovir ester, lamivudine, ribavirin, VX-960, Bo Saipuwei, GS-7977, tegobuvir, zanamivir, Oseltamivir, ganciclovir, FOSCARNET, acyclovir, azidothymidine AZT, Abacavir, Lopinavir, ritonavir or efavirenz.

31. pharmaceutical compositions according to any one of claims 1 to 30, it comprises supporting agent further.

32. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and PEG ylated compound that contain the oligonucleotide be made up of SEQ ID NO:3.

33. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and PEG ylated compound that contain the oligonucleotide be made up of SEQ ID NO:18.

34. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and PEG ylated compound that contain the oligonucleotide be made up of SEQ ID NO:11.

35. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:3.

36. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:18.

37. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:11.

38. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and Interferon Alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:3.

39. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and Interferon Alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:18.

40. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and Interferon Alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:11.

41. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and Pegylation thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:3.

42. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and Pegylation thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:18.

43. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and Pegylation thymosin α1 that contain the oligonucleotide be made up of SEQ ID NO:11.

44. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and glycol interferon alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:3.

45. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and glycol interferon alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:18.

46. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and glycol interferon alpha-2b that contain the oligonucleotide be made up of SEQ ID NO:11.

47. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and interferon lambda 1 that contain the oligonucleotide be made up of SEQ ID NO:3.

48. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and interferon lambda 1 that contain the oligonucleotide be made up of SEQ ID NO:18.

49. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and interferon lambda 1 that contain the oligonucleotide be made up of SEQ ID NO:11.

50. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and glycol interferon λ 1 that contain the oligonucleotide be made up of SEQ ID NO:3.

51. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and glycol interferon λ 1 that contain the oligonucleotide be made up of SEQ ID NO:18.

52. 1 kinds of pharmaceutical compositions, it comprises the ON chelate and glycol interferon λ 1 that contain the oligonucleotide be made up of SEQ ID NO:11.

53. for the preparation of a method for the pharmaceutical composition such as according to any one of claim 1 to 52, described method comprises:

54. for the preparation of a method for the pharmaceutical composition such as according to any one of claim 1 to 52, described method comprises:

At least one of b. adding gradually in the ON of described dissolving in pharmaceutically acceptable calcium and/or magnesium salt solution keeps solvable to make described ON chelate;

55. methods as described in claim 53 or 54, wherein said ON chelate solution and described polypeptide solution are facing the forward slip value using described pharmaceutical composition.

56. methods as described in claim 53 or 54, the ratio of the divalent metal salt wherein added in the ON of described dissolving is every 100mg oligonucleotide 0.1-50mg.

57. methods as described in claim 53 or 54, wherein said final ON concentration is 0.1-200mg/ml.

58. methods according to any one of claim 53 to 57, wherein said divalent metal salt is at least one in chloride salt, gluconate, citrate, lactate, malate, aspartate, fumarate, Ascorbate, benzoate, erythorbate, propionate, sulfate or bicarbonate.

59. methods according to any one of claim 53 to 57, wherein said divalent metal saline solution comprises at least one in calcium, magnesium, ferrum (2+), manganese, copper or zinc.

60. the method according to any one of claim 53 to 59, the described ON wherein used is selected from SEQ ID NO:1-18.

61. methods according to any one of claim 53 to 60, one or more polypeptide wherein said are selected from by the following group formed:

Thymosin α1;

Any alpha-interferon or its polyethylene glycol derivative;

Any beta-interferon or its polyethylene glycol derivative;

Any gamma interferon or its polyethylene glycol derivative;

Any λ-interferon or its polyethylene glycol derivative;

Intederon Alpha-2a or α-2b or α-N3;

Interferon beta-1a or β-1b;

Gamma interferon 1-b;

Interferon lambda 1 or λ 2 or λ 3;

PEG ylated compound or α-2b or λ 1 or λ 2 or λ 3;

Myrcludex B；

Any antiviral cell factor or its polyethylene glycol derivative;

Thymus protein A; With

62. 1 kinds of test kits, it comprises the pharmaceutical composition according to any one of claim 1 to 52.

63. test kits as claimed in claim 62, wherein said ON chelate and described at least one polypeptide are prepared respectively.

64. test kits as described in claim 63, wherein said ON chelate and the preparation of described at least one polypeptide are for jointly using by identical or different route of administration.